Therapeutic methods for prostate cancer

ABSTRACT

Therapeutic methods for the treatment of prostate cancer are described. The methods include a gene therapy method for prostate cancer using the BRCA family of genes, including the BRCA1 and BRCA2 genes. The BRCA family of gene products inhibit the growth and tumorigenesis of prostate cancer cells. Therapeutic methods using the BRCA family of gene products are also described.

GRANT STATEMENT

This invention was made in part from government support under Grant Numbers CA68485 and CA62161 from the National Institute of Health (NIH). The U.S. government has certain rights in the invention.

UTILITY STATEMENT

Both BRCA1 and BRCA2 proteins have been identified as inhibitors of the growth of mammalian prostate cancer cells. Thus, a nucleic acid segment encoding the BRCA1 protein and a nucleic acid segment encoding the BRCA2 protein can be used in gene therapy methods for the treatment of prostate cancer.

The discovery and purification of the BRCA1 and BRCA2 proteins has broad utility. The purified BRCA1 and BRCA2 proteins can be used in treating prostate cancer.

ACTIVITY STATEMENT

The BRCA1 gene product is an inhibitor of the growth and proliferation of mammalian prostate cancer cells. The BRCA1 gene product is a secreted protein, thus indicating that it acts on a receptor to produce this activity.

The BRCA2 gene product is an inhibitor of the growth and proliferation of mammalian prostate cancer cells. The BRCA2 protein is a secreted protein, thus indicating that it acts on a receptor to produce this activity.

TECHNICAL FIELD

The present invention relates to a therapy for prostate cancer; and more particularly to a gene therapy method for prostate cancer using the BRCA gene family, and still more particularly, using the BRCA1 gene.

The publications and other materials used herein to illuminate the background of the invention, and in particular cases, to provide additional details respecting the practice, are incorporated herein by reference, and for convenience, are referenced by author and date in the following text, and respectively group in the appended list of references.

Table of Abbreviations PPC-1 A prostate cancer cell line from primary tumor DU145 A prostate cancer cell line from brain metastasis LNCaP A prostate cancer cell line from lymph node metastasis PC3 A prostate cancer cell line from primary tumor TSU A prostate cancer cell line of unknown origin D17S855 A prostate cancer cell genotype, indicating number of alleles at markers flanking BRCA1 D17S1322 A prostate cancer cell genotype, indicating number of alleles at markers flanking BRCA1 D17S1327 A prostate cancer cell genotype, indicating number of alleles at markers flanking BRCA1 D17S1326 A prostate cancer cell genotype, indicating number of alleles at markers flanking BRCA1 D17S1325 A prostate cancer cell genotype, indicating number of alleles at markers flanking BRCA1 LXSN A retroviral vector derived from a mouse retrovirus Cys61Gly A mutation in the BRCA1 protein at amino acid 61 wherein a cysteine is substituted for a glycine 340stop A mutant BRCA1 protein wherein a stop codon is inserted in place of the codon coding for amino acid 340 del(343-1081) A BRCA1 mutant protein wherein amino acids 343-1081 have been deleted 1835stop A mutant BRCA1 protein wherein a stop codon has been inserted in place of the codon encoding the amino at position 1835 AIM V An animal serum free growth media for retroviral vectors PSA Prostate specific antigen LTR regulated gene expression controlled by the LXSN retroviral promoter

BACKGROUND ART

A staggering estimated 317,000 new cases of prostate cancer will be diagnosed and over 45,000 prostate cancer deaths will occur this year in the United States making prostate cancer the most frequently diagnosed and second leading cause of cancer mortality in men in the United States. Deaths from prostate cancer in the United States are increasing every year by 2%-3% because fewer men are dying from cardiovascular disease. (Walsh, 1994) Unfortunately, the age-specific mortality rate for prostate cancer continues to rise in spite of earlier detection by serum PSA or current prostate cancer treatment modalities. Moreover, at the time of diagnosis the majority of men will have prostate cancer at a stage for which there is no cure and the prognosis is dismal.

African-American men have the highest prostate cancer mortality rates of any population in the world, twice that of white men 65 years or older. Furthermore, survival rates in the United States for all stages of prostate cancer diagnosed between 1983 and 1990 was 81.3% for Whites, but only 66.4% for Blacks. Of all prostate cancer deaths in 1991, Blacks accounted for 15.8%, Hispanics for 2.5%, and American Indians, Chinese, and Japanese for less than 1%. The general United States population is 75% White, 12% African-American, 8% Hispanic, and 3% Asian.

The standard method of treatment for the past 50 years has been castration, surgical or chemical, but the prostate cancer has eventually become androgen-independent, resumed growth, and killed the patient. Clearly, better androgen blockade is not the answer for treating prostate cancer. Rather, treatment efforts should focus on modifying the mutations that lead to prostate oncogenesis. Although some genetic markers can at least partially predict patients who are likely to develop metastatic disease, it is still impossible to predict absolutely patient prognosis and response to therapy. (Walsh, 1994; Carter et al., 1990) Thus, even well implemented early detection programs may not completely eradicate the eventual development of metastasis in some patients.

The molecular biology of prostate cancer is poorly understood. Attempts to develop animal models of prostate cancer with transgenic mice have been less successful than for animal models of other cancers such as breast cancer. (Mulders et al., 1990; Oesterling, 1991; Jurincic et al., 1990; Hamdy et al., 1992; Pang et al., 1995; Matuo et al., 1989; Dodd et al., 1983; Greenberg et al., 1994; Greenberg et al, 1995; Tutrone et al., 1993; Matsui et al., 1990; Halter et al., 1992; Cato et al., 1989; Choi et al., 1987; Tutrone et al., 1993; Matsui et al., 1990; Halter et al., 1992; Muller et al., 1990) This has presumably happened because little is known about prostate-specific promoters and because study of oncogenes and tumor suppressor genes have yielded few clear-cut candidate genes for prostate cancer.

Inherited mutations in BRCA1, (Hall et al., 1990; Miki et al., 1994) confer lifetime risk of breast cancer greater than 80% and increased risk of ovarian cancer. (Newman et al., 1988; Ford et al., 1994). Multiple lines of evidence suggest that BRCA1 is a tumor suppressor for the following six reasons:

(1) Most (87%) inherited mutations truncate the BRCA1 protein, leading to loss of BRCA1 function. (Breast Cancer Information Core, 1996)

(2) The wild-type allele is lost from >90% of breast and ovarian tumors from patients with inherited BRCA1 mutations. (Friedman et al., 1994; Neuhausen et al., 1994; Smith et al., 1992)

(3) BRCA1 expression is reduced in breast and ovarian tumors from patients not selected for family history. (Thompson et al., 1995) In such tumors, somatic inactivation of BRCA1 may occur through mechanisms such as large deletions or epigenetic silencing of BRCA1 expression, rather than point mutation. (Futreal et al., 1994; Cropp et al., 1993; Saito et al., 1993; Cliby et al., 1993; Russell et al., 1990; Takahashi et al., 1995; Yang-Feng et al., 1993)

(4) Inhibition of BRCA1 expression with antisense oligonucleotides leads to accelerated growth of normal and malignant mammary epithelial cells. (Thompson et al., 1995)

(5) Overexpression of BRCA1 inhibits growth of breast and ovarian cancer cell lines derived from patients not selected for family history. (Holt et al., 1996)

(6) Transfection or infection of MCF-7 breast cancer cells with the wild type BRCA1 gene inhibits tumor development and suppresses growth of established tumors in nude mice. (Holt et al., 1996) The biochemical mechanism responsible for growth inhibition and tumor suppression by BRCA1 involves secretion, since BRCA1 has sequence homology and functional analogy to the granin protein family. Wild type BRCA1 is localized to the Golgi; (Jensen et al., 1996) and wild-type BRCA1 is also present in the nucleus, although reports differ in the relative amounts of nuclear versus cytoplasmic protein. (Chen et al., 1995)

There has been no affirmative suggestion of a treatment of prostatic cancer comprising a therapeutic application of the BRCA gene family, and particularly comprising a therapeutic application involving BRCA1. This is true despite certain epidemiological, genetic, and biological observations in the art, including the following four observations: (1) Breast and prostatic cancer, and ovarian and prostatic cancer, are associated in families, (Jishi et al., 1995; Anderson et al., 1993; Sellers et al., 1994; Tulinium et al., 1994) although the association is not observed in families in which index cases were patients with prostatic cancer (rather than breast or ovarian cancer). (Isaacs et al., 1995) (2) Inherited mutations in BRCA1 have been observed in prostatic cancer patients, both in families at high risk of breast and ovarian cancer (Ford et al., 1994; Friedman et al., 1994; Struewing et al., 1995) and in isolated patients. (Langston et al., 1996) (3) Prostatic tumors are frequently hemizygous for markers in or near BRCA1. (Williams et al., 1996; Gao et al., 1995; Brothman et al., 1995; Gao et al., 1995) (4) The malignant phenotype of the human prostatic cancer cell line PPC-1 was suppressed by transfer of an -30 Mb portion of chromosome 17 containing BRCA1. (Murakami et al., 1995). Additionally, although the 30 Mb portion of chromosome 17 contained BRCA1, it also contained numerous other genes and included a region proposed to contain a different tumor suppressor gene.

Given the prevalence of prostate cancer, what is needed, then, is an effective therapy for prostate cancer that addresses the disease at a molecular genetic level. Despite attempts to characterize the molecular biology of prostate cancer, such a therapy is lacking in the prior art.

DISCLOSURE OF THE INVENTION

A method to suppress the growth of a prostate tumor in a mammal is disclosed. The method comprises introducing to said tumor a vector comprising a nucleic acid sequence encoding a BRCA family gene product operatively linked to a promoter, wherein the production of the BRCA family gene product results in a decrease in the growth rate of the tumor. The vector can comprise a plasmid vector or a viral vector. Preferably, the vector comprises a retroviral vector. The prostate cancer can comprise gene-linked hereditary prostate cancer or sporadic prostate cancer.

A method to suppress the growth of a prostate tumor in a mammal wherein the method comprises introducing to said tumor a liposome complexed to a nucleic acid encoding a prostate tumor suppressing polypeptide operatively linked to a promoter, the nucleic acid encoding a BRCA family gene product, wherein production of the prostate tumor suppressing polypeptide results in a decrease of the growth rate of the tumor is also described.

The BRCA family gene product can comprise a BRCA1 targeted growth inhibitor agent or a BRCA2 targeted growth inhibitor agent, as defined herein. The BRCA family gene product can also comprise the BRCA1 gene product or the BRCA2 gene product, and nucleic acids encoding such products, as defined herein.

Therefore, an aspect of this invention concerns purified and isolated BRCA1 and BRCA2 gene products; and biologically functional and structural equivalents of each.

Another aspect of this invention is that the BRCA1 and BRCA2 gene products are tumor suppressor/growth inhibitors that exhibit tumor suppression/growth inhibition activity in prostate cancer.

Yet another aspect of this invention is that the BRCA1 and BRCA2 gene products are secreted, and thus, act on a receptor to impart their activity.

Important aspects of the present invention concern isolated DNA segments and recombinant vectors encoding the BRCA1 and the BRCA2 gene products, and the creation and use of recombinant host cells, through the application of recombinant DNA technology, which express the BRCA1 and BRCA2 gene products.

Introduction of Gene Products

Where the gene itself is employed to introduce the gene products, a convenient method of introduction will be through the use of a recombinant vector which incorporates the desired gene, together with its associated control sequences. The preparation of recombinant vectors is well known to those of skill in the art and described in many references, such as, for example, Sambrook et al. (1989), specifically incorporated herein by reference.

In vectors, it is understood that the DNA coding sequences to be expressed, in this case those encoding the tumor-suppressing gene products, are positioned adjacent to and under the control of a promoter. It is understood in the art that to bring a coding sequence under the control of such a promoter, one generally positions the 5′ end of the transcription initiation site of the transcriptional reading frame of the gene product to be expressed between about 1 and about 50 nucleotides “downstream” of (i.e., 3′ of) the chosen promoter. One may also desire to incorporate into the transcriptional unit of the vector an appropriate polyadenylation site (e.g., 5′-AATAAA-3′), if such a site was not contained within the original inserted DNA. Typically, these poly A addition sites are placed about 30 to 2000 nucleotides “downstream” of the coding sequence at a position prior to transcription termination.

While use of the control sequences of the specific gene (e.g., the BRCA1 promoter for BRCA1 and the BRCA2 promoter for BRCA2) will be preferred, there is no reason why other control sequences could not be employed, so long as they are compatible with the genotype of the cell being treated. Thus, one may mention other useful promoters by way of example, including, e.g., an SV40 early promoter, a long terminal repeat promoter from retrovirus, an actin promoter, a heat shock promoter, a metallothionein promoter, and the like.

For introduction of a BRCA family gene, such as BRCA1 and BRCA2, it is proposed that one will desire preferably to employ a vector construct that will deliver the desired gene to the affected cells. This will, of course, generally require that the construct be delivered to the targeted tumor cells, for example, prostate tumor cells. It is proposed that this may be achieved most preferably by introduction of the desired gene through the use of a viral vector to carry either BRCA family sequences efficiently to infect the tumor, or pretumorous tissue. These vectors will preferably be an adenoviral, a retroviral, a vaccinia viral vector or adeno-associated virus. These vectors are preferred because they have been successfully used to deliver desired sequences to cells and tend to have a high infection efficiency. An example of a particularly preferred vector is the LXSN retroviral vector described herein.

Commonly used viral promoters for expression vectors are derived from polyoma, cytomegalovirus, Adenovirus 2, and Simian Virus 40 (SV40). The early and late promoters of SV40 virus are particularly useful because both are obtained easily from the virus as a fragment which also contains the SV40 viral origin of replication. Smaller or larger SV40 fragments may also be used, provided there is included the approximately 250 bp sequence extending from the Hind III site toward the Bg1 I site located in the viral origin of replication. Further, it is also possible, and often desirable, to utilize promoter or control sequences normally associated with the desired gene sequence, provided such control sequences are compatible with the host cell systems.

The origin of replication may be provided either by construction of the vector to include an exogenous origin, such as may be derived from SV40 or other viral (e.g., Polyoma, Adeno, VSV, BPV) sources, or may be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter is often sufficient.

Definitions and Techniques Affecting Gene Products and Genes

The present invention concerns DNA segments, isolatable from mammalian tissue, which are free from genomic DNA and which are capable of conferring tumor suppressor/growth inhibitor activity in a recombinant host cell when incorporated into the recombinant host cell. As used herein, the term “mammalian tissue” refers to normal and cancerous mammalian breast, ovarian or prostate tissues, as exemplified by, but not limited to, HMEC, MCF-7 or PPC-1 cell lines. DNA segments capable of conferring tumor suppressor activity may encode complete BRCA1 and BRCA2 gene products, cleavage products and biologically actively functional domains thereof.

The term “BRCA family”, as used in the specification and in the claims, is contemplated to include the BRCA granins described herein, including BRCA1 and BRCA2 genes and gene products. The BRCA family is characterized by the tumor suppressor activity of the gene product and the granin box consensus sequence shown in FIG. 5.

The terms “BRCA1 gene product” and “BRCA1” or “BRCA2 gene product” and “BRCA2” as used in the specification and in the claims refer to proteins having amino acid sequences which are substantially identical to the native BRCA1 or BRCA2 amino acid sequences and which are biologically active in that they are capable of suppressing tumor growth or cross-reacting with an anti-BRCA1 or an anti-BRCA2 antibody raised against BRCA1 or BRCA2. Such sequences are disclosed, for example, by Miki et al. 1994 and Wooster et al. 1995. The terms “BRCA1 gene product” and “BRCA2 gene product” also include analogs of BRCA1 and BRCA2 molecules which exhibit at least some biological activity in common with native BRCA1 or BRCA2. Furthermore, those skilled in the art of mutagenesis will appreciate that other analogs, as yet undisclosed or undiscovered, may be used to construct BRCA1 or BRCA2 analogs. There is no need for a “BRCA1 gene product” or “BRCA1”, or a “BRCA2 gene product” or “BRCA2” to comprise all, or substantially all, of the amino acid sequence of the native BRCA1 or BRCA2 genes. Shorter or longer sequences are anticipated to be of use in the invention.

The terms “BRCA1 gene” and “BRCA2 gene” refer to any DNA sequence that is substantially identical to a DNA sequence encoding a BRCA1 gene product or a BRCA2 gene product as defined above. The terms also refer to RNA, or antisense sequences, compatible with such DNA sequences. A “BRCA1 gene” or a “BRCA2 gene” may also comprise any combination of associated control sequences.

The term “substantially identical”, when used to define either a BRCA1 or a BRCA2 amino acid sequence, or a BRCA1 or a BRCA2 nucleic acid sequence, means that a particular sequence, for example, a mutant sequence, varies from the sequence of natural BRCA1 or BRCA2 by one or more deletions, substitutions, or additions, the net effect of which is to retain at least some of biological activity of the BRCA1 or the BRCA2 protein. Alternatively, DNA analog sequences are “substantially identical” to specific DNA sequences disclosed herein if: (a) the DNA analog sequence is derived from coding regions of the natural BRCA1 or BRCA2 gene; or (b) the DNA analog sequence is capable of hybridization of DNA sequences of (a) under moderately stringent conditions and which encode biologically active BRCA1 or BRCA2; or (c) the DNA sequences are degenerative as a result of the genetic code to the DNA analog sequences defined in (a) and/or (b). Substantially identical analog proteins will be greater than about 80% to the corresponding sequence of the native protein. Sequences having lesser degrees of similarity but comparable biological activity are considered to be equivalents. In determining nucleic acid sequences, all subject nucleic acid sequences capable of encoding substantially similar amino acid sequences are considered to be substantially similar to a reference nucleic acid sequence, regardless of differences in codon sequences.

The term “BRCA1 targeted growth inhibitor agent”, as used in the specification and in the claims, is defined as the BRCA1 gene product characterized herein, whether isolated and purified directly from a natural source such as mammalian prostate, ovarian or breast cells, or produced using recombinant methods. The term “BRCA1 targeted growth inhibitor agent” also refers to a targeted growth inhibitor having the biological activity of tumor suppression and/or growth inhibition activity in mammalian prostate cancer cells. The term “BRCA1 targeted growth inhibitor agent” also refers to a targeted growth inhibitor agent which binds the BRCA1 receptor. The term “BRCA1 targeted growth inhibitor agent” also includes biologically functional equivalents of the BRCA1 gene product characterized herein, the term biologically functional equivalent defined herein to include, among others, proteins and protein fragments in which biologically functionally equivalent amino acids have been inserted, and peptidomimetics.

The term “BRCA2 targeted growth inhibitor agent” is used herein as “BRCA1 targeted growth inhibitor agent” above but applies to the BRCA2 gene product.

Percent Similarity

Percent similarity may be determined, for example, by comparing sequence information using the GAP computer program, available from the University of Wisconsin Geneticist Computer Group. The GAP program utilizes the alignment method of Needleman et al. 1970, as revised by Smith et al. 1981. Briefly, the GAP program defines similarity as the number of aligned symbols (i.e. nucleotides or amino acids) which are similar, divided by the total number of symbols in the shorter of the two sequences. The preferred default parameters for the GAP program include: (1) a unitary comparison matrix (containing a value of 1 for identities and 0 for non-identities) of nucleotides and the weighted comparison matrix of Gribskov et al., 1986, as described by Schwartz et al., 1979; (2) a penalty of 3.0 for each gap and an additional 0.01 penalty for each symbol and each gap; and (3) no penalty for end gaps.

The term “homology” describes a mathematically based comparison of sequence similarities which is used to identify genes or proteins with similar functions or motifs. Accordingly, the term “homology” is synonymous with the term “similarity” and “percent similarity” as defined above. Thus, the phrases “substantial homology” or “substantial similarity” have similar meanings.

Nucleic Acid Sequences

In certain embodiments, the invention concerns the use of tumor suppressor genes and gene products, such as the BRCA family gene products, including BRCA1 and BRCA2, that include within their respective sequences a sequence which is essentially that of a BRCA family gene, including the known BRCA1 and BRCA2 genes, or the corresponding proteins. The term “a sequence essentially as that of a BRCA family gene or gene product, including BRCA1 or BRCA2“, means that the sequence substantially corresponds to a portion of a BRCA family gene or gene product, including BRCA1 or BRCA2, and has relatively few bases or amino acids (whether DNA or protein) which are not identical to those of a BRCA family gene or gene product, including BRCA1 and BRCA2 (a biologically functional equivalent of, when referring to proteins). The term “biologically functional equivalent” is well understood in the art and is further defined in detail herein. Accordingly, sequences which have between about 70% and about 80%; or more preferably, between about 81% and about 90%; or even more preferably, between about 91% and about 99%; of amino acids which are identical or functionally equivalent to the amino acids of a BRCA family gene or gene product, including BRCA1 and BRCA2, will be sequences which are “essentially the same”.

BRCA1 and BRCA2 genes which have functionally equivalent codons are also covered by the invention. The term “functionally equivalent codon” is used herein to refer to codons that encode the same amino acid, such as the six codons for arginine or serine, and also to refer to codons that encode biologically equivalent amino acids (see FIG. 2).

It will also be understood that amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids or 5′ or 3′ sequences, and yet still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein activity where protein expression is concerned. The addition of terminal sequences particularly applies to nucleic acid sequences which may, for example, include various non-coding sequences flanking either of the 5′ or 3′ portions of the coding region or may include various internal sequences, i.e., introns, which are known to occur within genes.

The present invention also encompasses the use of DNA segments which are complementary, or essentially complementary, to the sequences set forth in the specification. Nucleic acid sequences which are ” complementary” are those which are base-pairing according to the standard Watson-Crick complementarity rules. As used herein, the term “complementary sequences” means nucleic acid sequences which are substantially complementary, as may be assessed by the same nucleotide comparison set forth above, or as defined as being capable of hybridizing to the nucleic acid segment in question under relatively stringent conditions such as those described herein.

Nucleic acid hybridization will be affected by such conditions as salt concentration, temperature, or organic solvents, in addition to the base composition, length of the complementary strands, and the number of nucleotide base mismatches between the hybridizing nucleic acids, as will be readily appreciated by those skilled in the art. Stringent temperature conditions will generally include temperatures in excess of 30° C., typically in excess of 37° C., and preferably in excess of 45° C. Stringent salt conditions will ordinarily be less than 1,000 mM, typically less than 500 mM, and preferably less than 200 mM. However, the combination of parameters is much more important than the measure of any single parameter. (See, e.g., Wetmur & Davidson, 1968; Kanehisa, 1984).

Probe sequences may also hybridize specifically to duplex DNA under certain conditions to form triplex or other higher order DNA complexes. The preparation of such probes and suitable hybridization conditions are well known in the art.

As used herein, the term “DNA segment” refers to a DNA molecule which has been isolated free of total genomic DNA of a particular species. Furthermore, a DNA segment encoding a BRCA1 gene product or encoding a BRCA2 gene product refers to a DNA segment which contains BRCA1 coding sequences or contains BRCA2 coding sequences, yet is isolated away from, or purified free from, total genomic DNA of Homo sapiens. Included within the term “DNA segment” are DNA segments and smaller fragments of such segments, and also recombinant vectors, including, for example, plasmids, cosmids, phages, viruses, and the like.

Similarly, a DNA segment comprising an isolated or purified BRCA1 gene or BRCA2 gene refers to a DNA segment including BRCA1 coding sequences substantially away from other naturally occurring genes or protein encoding sequences or including BRCA2 coding sequences isolated substantially away from other naturally occurring genes or protein encoding sequences. In this respect, the term “gene” is used for simplicity to refer to a functional protein, polypeptide or peptide encoding unit. As will be understood by those in the art, this functional term includes both genomic sequences and cDNA sequences. “Isolated substantially away from other coding sequences” means that the gene of interest, in this case, the BRCA1 gene or the BRCA2 gene, forms the significant part of the coding region of the DNA segment, and that the DNA segment does not contain large portions of naturally-occurring coding DNA, such as large chromosomal fragments or other functional genes or CDNA coding regions. of course, this refers to the DNA segment as originally isolated, and does not exclude genes or coding regions later added to the segment by the hand of man.

In particular embodiments, the invention concerns isolated DNA segments and recombinant vectors incorporating DNA sequences which encode a BRCA1 protein that includes within its amino acid sequence the amino acid sequence of SEQ ID NO:2. In other particular embodiments, the invention concerns isolated DNA segments and recombinant vectors incorporating DNA sequences which encode a protein that includes within its amino acid sequence the amino acid sequence of the BRCA1 protein corresponding to human prostate tissue.

In particular embodiments, the invention concerns isolated DNA segments and recombinant vectors incorporating DNA sequences which encode a BRCA2 protein that includes within its amino acid sequence the amino acid sequence of SEQ ID NO:4. In other particular embodiments, the invention concerns isolated DNA segments and recombinant vectors incorporating DNA sequences which encode a protein that includes within its amino acid sequence the amino acid sequence of the BRCA2 protein corresponding to human prostate tissue.

It will also be understood that this invention is not limited to the particular nucleic acid and amino acid sequences of SEQ ID NOS:1, 2, 3 and 4. Recombinant vectors and isolated DNA segments may therefore variously include the BRCA1 and BRCA2 encoding regions themselves, include coding regions bearing selected alterations or modifications in the basic coding region, or include encoded larger polypeptides which nevertheless include BRCA2 or BRCA2 encoding regions or may encode biologically functional equivalent proteins or peptides which have variant amino acid sequences.

In certain embodiments, the invention concerns isolated DNA segments and recombinant vectors which encode a protein or peptide that includes within its amino acid sequence an amino acid sequence essentially as set forth in SEQ ID NO:2 or SEQ ID NO:4, and methods of treating prostate cancer using these DNA segments. Naturally, where the DNA segment or vector encodes a full length BRCA1 or BRCA2 gene product, the most preferred sequences are those which are essentially as set forth in SEQ ID NO:1 and SEQ ID NO:3 and which encode a protein that exhibits tumor suppressor activity in human prostate cancer cells, as may be determined by the prostate cancer cell growth inhibition experiments, as disclosed herein.

The term “a sequence essentially as set forth in SEQ ID NO:2” means that the sequence substantially corresponds to a portion of SEQ ID NO:2 and has relatively few amino acids which are not identical to, or a biologically functional equivalent of, the amino acids of SEQ ID NO:2. The term “biologically functional equivalent” is well understood in the art and is further defined in detail herein. Accordingly, sequences, which have between about 70% and about 80%; or more preferably, between about 81% and about 90%; or even more preferably, between about 91% and about 99%; of amino acids which are identical or functionally equivalent to the amino acids of SEQ ID NO:2, will be sequences which are “essentially as set forth in SEQ ID NO:2”. The term “a sequence essentially set forth in SEQ ID NO:4” has a similar meaning.

In particular embodiments, the invention concerns gene therapy methods that use isolated DNA segments and recombinant vectors incorporating DNA sequences which encode a protein that includes within its amino acid sequence an amino acid sequence in accordance with SEQ ID NO:2 or in accordance with SEQ ID NO:4, SEQ ID NO:2 and SEQ ID NO:4 derived from prostate tissue from Homo sapiens. In other particular embodiments, the invention concerns isolated DNA sequences and recombinant DNA vectors incorporating DNA sequences which encode a protein that includes within its amino acid sequence the amino acid sequence of the BRCA1 protein from human prostate tissue, or which encode a protein that includes within its amino acid sequence the amino acid sequence of the BRCA2 protein from human prostate tissue.

In certain other embodiments, the invention concerns isolated DNA segments and recombinant vectors that include within their sequence a nucleic acid sequence essentially as set forth in SEQ ID NO:1 , or a nucleic acid sequence essentially as set forth in SEQ ID NO:3, and methods of treating prostate cancer using these sequences. The term “essentially as set forth in SEQ ID NO:1” is used in the same sense as described above and means that the nucleic acid sequence substantially corresponds to a portion of SEQ ID NO:1, respectively, and has relatively few codons which are not identical, or functionally equivalent, to the codons of SEQ ID NO:1 , respectively. Again, DNA segments which encode gene products exhibiting tumor suppression activity of the BRCA1 and BRCA2 gene products will be most preferred. The term “functionally equivalent codon” is used herein to refer to codons that encode the same amino acid, such as the six codons for arginine or serine, and also to refer to codons that encode biologically equivalent amino acids (see FIG. 2). The term “essentially as set forth in SEQ ID NO:3” has a similar meaning.

The nucleic acid segments of the present invention, regardless of the length of the coding sequence itself, may be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol. For example, nucleic acid fragments may be prepared which include a short stretch complementary to SEQ ID NO:1 or SEQ ID NO:3, such as about 10 nucleotides, and which are up to 10,000 or 5,000 base pairs in length, with segments of 3,000 being preferred in certain cases. DNA segments with total lengths of about 1,000, 500, 200, 100 and about 50 base pairs in length are also contemplated to be useful.

The DNA segments of the present invention encompass biologically functional equivalent BRCA1 and BRCA2 proteins and peptides. Such sequences may rise as a consequence of codon redundancy and functional equivalency which are known to occur naturally within nucleic acid sequences and the proteins thus encoded. Alternatively, functionally equivalent proteins or peptides may be created via the application of recombinant DNA technology, in which changes in the protein structure may be engineered, based on considerations of the properties of the amino acids being exchanged. Changes designed by man may be introduced through the application of site-directed mutagenesis techniques, e.g., to introduce improvements to the antigenicity of the protein or to test BRCA1 and BRCA2 mutants in order to examine tumor suppression activity at the molecular level.

If desired, one may also prepare fusion proteins and peptides, e.g., where the BRCA1 or BRCA2 coding regions are aligned within the same expression unit with other proteins or peptides having desired functions, such as for purification or immunodetection purposes (e.g., proteins which may be purified by affinity chromatography and enzyme label coding regions, respectively).

Recombinant vectors form important further aspects of the present invention. Particularly useful vectors are contemplated to be those vectors in which the coding portion of the DNA segment is positioned under the control of a promoter. The promoter may be in the form of the promoter which is naturally associated with the BRCA1 or BRCA2 gene(s), e.g., in prostate cancer cells, as may be obtained by isolating the 5′ non-coding sequences located upstream of the coding segment or exon, for example, using recombinant cloning and/or PCR technology, in connection with the compositions disclosed herein.

In other embodiments, it is contemplated that certain advantages will be gained by positioning the coding DNA segment under the control of a recombinant, or heterologous, promoter. As used herein, a recombinant or heterologous promoter is intended to refer to a promoter that is not normally associated with a BRCA1 or BRCA2 gene in its natural environment. Such promoters may include promoters isolated from bacterial, viral, eukaryotic, or mammalian cells. Naturally, it will be important to employ a promoter that effectively directs the expression of the DNA segment in the cell type chosen for expression. The use of promoter and cell type combinations for protein expression is generally known to those of skill in the art of molecular biology, for example, see Sambrook et al., 1989, specifically incorporated herein by reference. The promoters employed may be constitutive, or inducible, and can be used under the appropriate conditions to direct high level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins or peptides. Appropriate promoter systems contemplated for use in high-level expression include, but are not limited to, the LXSN promoter, which is more fully described below.

As mentioned above, in connection with expression embodiments to prepare recombinant BRCA1 and BRCA2 proteins and peptides, it is contemplated that longer DNA segments will most often be used, with DNA segments encoding the entire BRCA1 or BRCA2 protein, functional domains or cleavage products thereof, being most preferred. However, it will be appreciated that the use of shorter DNA segments to direct the expression of BRCA1 and BRCA2 peptides or epitopic core regions, such as may be used to generate anti-BRCA1 or anti-BRCA2 antibodies, also falls within the scope of the invention.

DNA segments which encode peptide antigens from about 15 to about 50 amino acids in length, or more preferably, from about 15 to about 30 amino acids in length are contemplated to be particularly useful. DNA segments encoding peptides will generally have a minimum coding length in the order of about 45 to about 150, or to about 90 nucleotides. DNA segments encoding full length proteins may have a minimum coding length on the order of about 5,600 nucleotides for a protein in accordance with SEQ ID NO:2 or a minimum coding length on the order of about 10,300 nucleotides for a protein in accordance with SEQ ID NO:4.

Naturally, the present invention also encompasses DNA segments which are complementary, or essentially complementary, to the sequence set forth in SEQ ID NO:1 or the sequence set forth in SEQ ID NO:3. The terms “complementary” and “essentially complementary” are defined above. Excepting intronic or flanking regions, and allowing for the degeneracy of the genetic code, sequences which have between about 70% and about 80%; or more preferably, between about 81% and about 90%; or even more preferably, between about 91% and about 99%; of nucleotides which are identical or functionally equivalent (i.e. encoding the same amino acid) of nucleotides of SEQ ID NO:1 or to the nucleotides of SEQ ID NO:3, will be respectively sequences which are “essentially as set forth in SEQ ID NO:1” and will be sequences which are “essentially as set forth in SEQ ID NO:3”. Sequences which are essentially the same as those set forth in SEQ ID NO:1 or as those set forth in SEQ ID NO:3 may also be functionally defined as sequences which are capable of hybridizing to a nucleic acid segment containing the complement of SEQ ID NO:1 or to a nucleic acid segment containing the complement of SEQ ID NO:3 under relatively stringent conditions. Suitable relatively stringent hybridization conditions are described herein and will be well known to those of skill in the art (Sambrook et al., 1989).

Biological Functional Equivalent Proteins and Peptides

Modification and changes may be made in the structure of the BRCA1 protein and the BRCA2 protein, or in cleavage products of these proteins, and still obtain a molecule having like or otherwise desirable characteristics. For example, certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules or receptors, specifically the BRCA1 or BRCA2 receptor. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence (or, of course, its underlying DNA coding sequence) and nevertheless obtain a protein with like (agonistic) properties. Equally, the same considerations may be employed to create a protein or polypeptide with countervailing (e.g., antagonistic) properties. It is thus contemplated by the inventors that various changes may be made in the sequence of the BRCA1 and BRCA2 proteins or peptides (or underlying DNA) without appreciable loss of their biological utility or activity.

Two designations for amino acids are used interchangeably throughout this application, as is common practice in the art. Alanine=Ala (A); Arginine=Arg (R); Aspartate=Asp (D); Asparagine=Asn (N); Cysteine=Cys (C); Glutamate=Glu (E); Glutamine=Gln (Q); Glycine=Gly (G); Histidine=His (H); Isoleucine=Ile (I); Leucine=Leu (L); Lysine=Lys (K); Methionine=Met (M); Phenylalanine=Phe (F); Proline=Pro (P); Serine=Ser (S); Threonine=Thr (T); Tryptophan=Trp (W); Tyrosine=Tyr (Y); Valine=Val (V).

It is also well understood by the skilled artisan that, inherent in the definition of a biologically functional equivalent protein or peptide, is the concept that there is a limit to the number of changes that may be made within a defined portion of the molecule and still result in a molecule with an acceptable level of equivalent biological activity. Biologically functional equivalent peptides are thus defined herein as those peptides in which certain, not most or all, of the amino acids may be substituted. Of course, a plurality of distance proteins/peptides with different substitutions may easily be made and used in accordance with this invention.

It is also well understood that where certain residues are shown to be particularly important to the biological or structural properties of a protein or peptide, e.g., residues in active sites, such residues may not generally be exchanged. This is the case in the present invention where an exchange in the granin box domain may alter the fact that the BRCA1 and BRCA2 proteins are secreted.

Amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. An analysis of the size, shape and type of the amino acid side-chain substituents reveals that arginine, lysine, and histidine are all positively charged residues; that alanine, glycine and serine are all a similar size; and that phenylalanine, tryptophan and tyrosine all have a generally similar shape. Therefore, based upon these considerations, arginine, lysine and histidine are defined herein as biologically functional equivalents of each other; alanine, glycine and serine are defined herein as biologically functional equivalents of each other; and phenylalanine, tryptophan and tyrosine are defined herein as biologically functional equivalents of each other.

In making such changes, the hydropathic index of amino acids may be considered. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics. These indices are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cystein/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine (−4.5).

The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte & Doolittle, 1982, incorporated herein by reference) . It is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. In making changes based upon the hydropathic index, the substitution of amino acids whose hydropathic indices are within ±1 are particularly preferred, and those with ±2 are more particularly preferred, those which are within ±0.5 are even more particularly preferred.

It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity, particularly where the biological functional equivalent protein or peptide thereby created is intended for use in immunological embodiments. U.S. Pat. No. 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e., with a biological property of the protein. It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ±1); glutamate (+3.0 ±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline (−0.5 ±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4).

In making changes based upon similar hydrophilicity values, the substitution of amino acids that shows hydrophilicity values are within ±2 is preferred, those which are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

As outlined above, amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions which take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

While discussion has focused on functionally equivalent polypeptides arising from amino acid changes, it will be appreciated that these changes may be effected by alteration of the encoding DNA, taking into consideration also that the genetic code is degenerate and that two or more codons may code for the same amino acid.

Sequence Modification Techniques

Modifications to the BRCA1 and BRCA2 peptides may be carried out using techniques such as site directed mutagenesis. Site-specific mutagenesis is a technique useful in the preparation of individual peptides, or biological functional equivalent proteins or peptides, through specific mutagenesis of the underlying DNA. The technique further provides a ready ability to prepare and to test sequence variants, for example, incorporating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the DNA. Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA sequence of desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed. Typically, a primer of about 17 to 25 nucleotides in length is preferred, with about 5 to 10 residues on both sides of the junction of the sequence being altered.

In general, the technique of site-specific mutagenesis is well known in the art as exemplified by publications (Adelman et al. 1983). As will be appreciated, the technique typically employs a phage vector which exists in both a single stranded and double stranded form. Typical vectors useful in site-directed mutagenesis include vectors such as the M13 phage (Messing et al., 1981). These phage vectors are readily commercially available and their use is generally well known to those skilled in the art. Double stranded plasmids are also routinely employed in site directed mutagenesis which eliminates the step of transferring the gene of interest from a plasmid to a phage.

In general, site-directed mutagenesis in accordance herewith is performed by first obtaining a single stranded vector or melting apart the two strands of a double stranded vector which includes within its sequence a DNA sequence which encodes a BRCA family gene, including BRCA1 and/or BRCA2. An oligonucleotide primer bearing the desired mutated sequence is prepared, generally synthetically, for example by the method of Crea et al. (1978). This primer is then annealed with the single stranded vector, and subjected to DNA polymerizing enzymes such as E. Coli polymerase I Klenow fragment, in order to complete the synthesis of the mutation-bearing strand. Thus, a heteroduplex is formed wherein one strand encodes the original non-mutated sequence and the second strand bears the desired mutation. This heteroduplex vector is then used to transform appropriate cells, such as E. Coli cells, and clones are selected which include recombinant vectors bearing the mutated sequence arrangement.

The preparation of sequence variants of the selected gene using site-directed mutagenesis is provided as a means of producing potentially useful BRCA1, BRCA2 or other BRCA family species and is not meant to be limiting as there are other ways in which sequence variants of these peptides may be obtained. For example, recombinant vectors encoding the desired genes may be treated with mutagenic agents to obtain sequence variants (see, e.g., a method described by Eichenlab, 1979) for the mutagenesis of plasmid DNA using hydroxylamine.

Other Structural Equivalents

In addition to the peptidyl compounds described herein, the inventors also contemplate that other sterically similar compounds may be formulated to mimic the key portions of the peptide structure. Such compounds, which may be termed peptidomimetics, may be used in the same manner as the peptides of the invention and hence are also functional equivalents. The generation of a structural functional equivalent may be achieved by the techniques of modeling and chemical design known to those of skill in the art. It will be understood that all such sterically similar constructs fall within the scope of this invention.

Accordingly, it is an object of this invention to provide a gene therapy for prostate cancer which includes the BRCA gene family, and particularly includes the BRCA1 gene.

It is a further object of this invention to provide a therapy for prostate cancer that addresses the disease at a molecular genetic level.

It is a further object of this invention to provide a method of preventing prostate cancer comprising prophylactic gene therapy using the BRCA gene family, and particularly the BRCA1 gene.

Some of the objects of the invention having been stated hereinabove, other objects will become evident as the description proceeds, when taken in connection with the accompanying Laboratory Examples and drawings as best described hereinbelow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A presents a graphical depiction of the growth of PPC-1 tumors treated with the vector alone.

FIG. 1B presents a graphical depiction of PPC-1 tumors treated with BRCA1. In both FIGS. 1A and 1B the diamond shapes represent day 0, the solid square shapes represent day 16, and the triangle shapes represent day 26.

FIG. 2 is a table of the genetic code.

FIG. 3 is a diagram showing structural features of the human BRCA1 protein [SEQ ID NO:2] covering 864 amino acids.

FIG. 4 is a diagram showing sequence alignment of the granin region of selected granin family members compared with BRCA1.

FIG. 5 is a diagram showing sequence alignment of the granin region of selected granin family members compared with BRCA1 and BRCA2.

FIGS. 6A-6D depict the sequence of the BRCA1 gene [SEQ ID NO:1].

FIGS. 7A-7F depict the sequence of the BRCA2 gene [SEQ ID NO:3].

FIGS. 8A-8C depict the sequence of the BRCA2 protein [SEQ ID NO:4].

DETAILED DESCRIPTION OF THE INVENTION

For the purposes of the subsequent description, the following definitions will be used:

Nucleic acid sequences which are “complementary” are those which are capable of base-pairing according to standard Watson-Crick complementarity rules. That is, that the larger purines will always base pair with the smaller pyrimidines to form only combinations of Guanine paired with Cytosine (G:C) and Adenine paired with either Thymine (A:T) in the case of DNA, or Adenine paired with Uracil (A:U) in the case of RNA.

“Hybridization techniques” refer to molecular biological techniques which involve the binding or hybridization of a probe to complementary sequences in a polynucleotide. Included among these techniques are northern blot analysis, southern blot analysis, nuclease protection assay, etc.

“Hybridization” and “binding” in the context of probes and denatured DNA are used interchangeably. Probes which are hybridized or bound to denatured DNA are aggregated to complementary sequences in the polynucleotide. Whether or not a particular probe remains aggregated with the polynucleotide depends on the degree of complementarity, the length of the probe, and the stringency of the binding conditions. The higher the stringency, the higher must be the degree of complementarity and/or the longer the probe.

“Probe” refers to an oligonucleotide or short fragment of DNA designed to be sufficiently complementary to a sequence in a denatured nucleic acid to be probed and to be bound under selected stringency conditions.

“Label” refers to a modification to the probe nucleic acid that enables the experimenter to identify the labeled nucleic acid in the presence of unlabeled nucleic acid. Most commonly, this is the replacement of one or more atoms with radioactive isotopes. However, other labels include covalently attached chromophores, fluorescent moieties, enzymes, antigens, groups with specific reactivity, chemiluminescent moieties, and electrochemically detectable moieties, etc.

“Tissuemizer” describes a tissue homogenization probe.

“PCR technique” describes a method of gene amplification which involves sequenced-based hybridization of primers to specific genes within a DNA sample (or library) and subsequent amplification involving multiple rounds of annealing, elongation and denaturation using a heat-stable DNA polymerase. Such techniques are described in U.S. Pat. No. 4,683,202, the contents of which are herein incorporated by reference.

“RT-PCR” is an abbreviation for reverse transcriptase-polymerase chain reaction. Subjecting mRNA to the reverse transcriptase enzyme results in the production of CDNA which is complementary to the base sequences of the mRNA. Large amounts of selected cDNA can then be produced by means of the polymerase chain reaction which relies on the action of heat-stable DNA polymerase produced by Thermus aquaticus for its amplification action.

“Nucleus protection assay” refers to a method of RNA quantification which employs strand specific nucleuses to identify specific RNAs by detection of duplexes.

“In situ hybridization of RNA” refers to the use of labeled DNA probes employed in conjunction with histological sections on which RNA is present and with which the labeled probe can hybridize allowing an investigator to visualize the location of the specific RNA within the cell.

“Cloning” describes separation and isolation of single genes.

“Sequencing” describes the determination of the specific order of nucleic acids in a gene or polynucleotide.

The term “cleavage product” is defined as a polypeptide fragment produced from the targeted growth inhibitor described above by natural proteolytic processes. Preferably such a cleavage product will have biological activity including, but not limited to, tumor suppression and/or growth inhibition activity in mammalian prostate cancer cells. This term also includes such polypeptide fragments when produced via recombinant techniques and also includes biological functional equivalents of such fragments, the term biologically functional equivalent defined herein to include, among others, proteins in which biologically functionally equivalent amino acids have been inserted, and peptidomimetics.

The term “granin box domain” is defined as the consensus granin box domain of amino acids set forth in FIGS. 3 and 5.

The term “recombinant host cell” is defined as a single cell or multiple cells within a cell line which are capable of undergoing genetic manipulation through well-known and art recognized techniques of transformation, transfection, transduction and the like. Examples of contemplated recombinant host cells include, but are not limited to, cell lines derived from normal or cancerous mammalian prostate, breast or ovarian tissue, other eukaryotic cells, and microorganisms. Specific examples of recombinant host cells described herein include PPC-1 cells.

The phrase “operably linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. For instance, a promoter is operably linked to a coding sequence if the promoter affects its transcription or expression.

The practice of the present invention employs, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA, genetics, and immunology. (See, e.g., Maniatis et al., 1982; Sambrook et al. , 1989; Ausubel et al., 1992; Glover, 1985; Anand, 1992; Guthrie & Fink, 1991).

Construction of Retroviral Vectors

Viral vectors containing a DNA sequence that encodes for a protein having an amino acid sequence as essentially set forth in SEQ ID NO:2 are constructed using techniques that are well known in the art. This sequence includes the BRCA1 gene product. Viral vectors containing a DNA sequence essentially set forth in SEQ ID NO:1 (the BRCA1 gene) can also be constructed using techniques that are well known in the art. See Sambrook et al., 1989 or Ausubel et al., 1992. Retroviral vectors such as the LXSN vector described herein, adenoviral vectors, or adeno-associated viral vectors are all useful methods for delivering genes into prostate cancer cells. The viral vector is constructed by cloning the DNA sequence as essentially set forth in SEQ ID NO:1 into a retroviral vector such as a prostate selective vector. Most preferably, the full length (coding region) cDNA for BRCA1 is cloned into the retroviral vector. The retroviral vector is then transfected into virus producing cells in the following manner: Viruses are prepared by transfecting PA317 cells with the retroviral vector DNAs which are purified in Wong et al., 1988. Following transfection, the PA317 cells are split and then treated with G418 until individual clones can be identified and expanded. Each clone is then screened for its titer by analyzing its ability to transfer G418 resistance (since the retroviral vector contains a Neomycin® resistance gene). The clones which have the highest titer are then frozen in numerous aliquots and tested for sterility, presence of replication-competent retrovirus, and presence of mycoplasma. Methods generally employed for construction and production of retroviral vectors have been described above and in Miller et al., 1990.

Once high titer viral vector producing clones are identified, then patients with prostate cancer are treated as described below.

It will be apparent to one having ordinary skill in the art that different length DNA segments encoding a BRCA family gene product can be cloned into the retroviral vectors. Well-known techniques such as restriction enzyme digests can be used to select sequences having lengths of particular interest. Moreover, the data more fully described herein characterizes appropriate sequence lengths. For example, the sequence of BRCA1 representing a splice variant encoding amino acids 72-1863 is a particularly useful one for growth inhibition studies of cancer cells including human prostate cancer cells.

Pharmaceutical Compositions

In a preferred embodiment, the present invention provides pharmaceutical compositions comprising a polypeptide or polynucleotide of the present invention and a physiologically acceptable carrier. More preferably, a pharmaceutical composition comprises a BRCA family polypeptide or a polynucleotide that encodes those polypeptides.

A composition of the present invention is typically administered parenterally in dosage unit formulations containing standard, well-known nontoxic physiologically acceptable carriers, adjuvants, and vehicles as desired. The term “parenteral” as used herein includes intravenous, intra-muscular, intraarterial injection, or infusion techniques.

Injectable preparations, for example sterile injectable aqueous or oleaginous suspensions, are formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol.

Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or di-glycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

Preferred carriers include neutral saline solutions buffered with phosphate, lactate, Tris, and the like. Of course, one purifies the vector sufficiently to render it essentially free of undesirable contaminants, such as defective interfering adenovirus particles or endotoxins and other pyrogens such that it does not cause any untoward reactions in the individual receiving the vector construct. A preferred means of purifying the vector involves the use of buoyant density gradients, such as cesium chloride gradient centrifugation.

A transfected cell can also serve as a carrier. By way of example, a liver cell can be removed from an organism, transfected with a polynucleotide of the present invention using methods set forth above and then the transfected cell returned to the organism (e.g. injected intravascularly).

The prostate cancer susceptibility/tumor suppressor gene BRCA1 has a role in prostate cancer. Prostatic cancer cell lines that have lost expression of BRCA1 protein are inhibited by transfection of wild-type BRCA1, and small human prostate tumors established in mice are inhibited by injection of a retroviral vector expressing wild-type BRCA1.

An in vivo retroviral vector-mediated gene therapy for the treatment of advanced prostate cancer is also described. Prostate cancer provides a model system in which a retroviral vector is employed to direct gene transfer effects toward the malignant cells without producing expression in the nearby non-dividing cells. The use of retroviral vectors provides specificity since only cancer cells within the areas of injection are expected to express the LTR-regulated BRCA1 genes. Therefore, the likelihood of selective gene transfer to the tumor cells is enhanced. The uptake and expression of the viral vectors can be readily assessed in these model systems because these cells are readily accessible for pathologic, biochemical, and molecular analysis.

Gene therapy is the direct transfer of engineered DNA into diseased cells for the purpose of therapy. The gene therapy approach taken herein initially targets those patients who have end-stage prostate cancer and who have failed chemotherapy. Human gene therapy is facilitated by concurrent advances, particularly in the past five years, both in the molecular biology of vectors for recombinant DNA transfer and in the development of research strategies using therapeutic gene transfer in animal models of disease. Gene therapy approaches have been shown to be successful in numerous animal models.

The following examples are set forth to illustrate the subject invention. The examples should not be considered as limiting, the scope of the invention being defined by the claims appended hereto.

EXAMPLE 1 Wild Type BRCA1 Suppresses Cell Growth in Prostate Cell Lines with Low Expression of BRCA1 Protein

In order to assess the effects of BRCA1 overexpression on cell growth, prostate cancer cell lines, PPC-1, DU145, LNCaP, PC3, and TSU, were transfected with wild-type and mutant BRCA1 genes. A Southern blot demonstrated transfer of the vector into transfected cell lines and tumors. Cell lines were characterized for BRCA1 at genomic, transcript, and protein expression levels. Genotypes of D17S855, D17Sl322, D17S1327, D17S1326, and D17S1325 suggest that both alleles of BRCA1 are present in DU145, but that the other cell lines have lost one BRCA1 allele (Table 1). Levels of BRCA1 protein detectable by Western blot varied from none in PPC-1 and LNCaP to moderate in TSU.

Wild-type BRCA1 inhibited the growth of prostate cancer cell lines PPC-1, LNCaP, and DU145 (Table 1). Mutant BRCA1 constructs, whether RING finger missense, 5′ or 3′ truncation, or in-frame deletion, did not inhibit growth. Cell lines PC3 and TSU were not inhibited by BRCA1. The resistant lines express moderate or high levels of BRCA1 protein on Western blot, despite apparent 10 hemizygosity at the BRCA1 locus.

TABLE 1 Effect of BRCA1 expression vectors on growth of prostate cancer cells PPC-1 LNCaP DU145 PC3 TSU BRCA1 genotype in LXSN-BRCA1 vector wildtype  0 + 0.3  1 + 0.3  4 + 1.4 73 + 3.9 130 + 4.5 Cys61Gly 79 + 0.6 41 + 2.2 70 + 2.0 73 + 2.7 139 + 5.5 340stop 67 + 3.4 32 + 1.7 61 + 1.1 67 + 2.8 130 + 2.4 del(343- 61 + 3.2 28 + 0.8 57 + 3.4 70 + 2.6 141 + 6.3 1081) 1835stop 65 + 3.6 28 + 1.7 57 + 2.9 72 + 1.4 134 + 8.7 Number of alleles at markers flanking BRCA1 D17855 1 1 1 1 D17S1322 1 2 1 1 D17S1327 1 2 1 1 D17S1326 1 2 1 1 D17S1325 1 2 1 1 BRCA1 + + + + transcript BRCA1 0 0 + + ++ protein on Western blot Source of primary lymph brain primary ? cells tumor node meta- tumor meta- stasis stasis

EXAMPLE 2 In vivo Transduction of Established PPC-1 Tumors by LXSN-BRCA1 in Nude Mice Slows Tumor Growth and Induces Tumor Regression

LXSN-BRCA1 vectors were injected into established PPC-1 tumors to determine if wild-type BRCA1 could be integrated into tumor cells and inhibit tumor growth. The PPC-1 cell line was selected for tumor suppression studies in animals because it is derived from a primary prostatic cancer and because it forms reproducible and measurable flank tumors in mice. In the first experiment, 11 tumors were injected with the LXSN-BRCA1, 11 tumors with the parent vector LXSN, and 6 tumors with media alone (Table 2). When results of the first experiment suggested that initial tumor size might influence inhibition by BRCA1, 5 additional tumors were treated with BRCA1. Each treatment group included tumors ranging in size from 0.5 mm³ to >5 mm³. Gene transfer of the retroviral vector was demonstrated by Southern blot of cell lines and injected tumors. The vector could be detected in 20-40% of injected tumors.

Rate of tumor growth between day 0 and day 26 was measured by linear regression of tumor size on time after first treatment. Tumor growth was significantly different for mice treated with BRCA1 compared to either those treated with vector alone (p<0.0001) or media alone (p<0.0001). BRCA1 treatment significantly inhibited growth for tumors of all initial sizes, but the effect was most pronounced for the smallest tumors (<2 mm³ at day 0) (P<0.00001). Small tumors were much more responsive to the single BRCA1 treatment than were larger tumors. All BRCA1 -treated tumors that were initially<2 mm³ disappeared completely. Of tumors with initial masses of 2 to 5 mm³ treated with BRCA1, two disappeared entirely, one decreased in size, and two grew substantially. The four tumors treated with BRCA1 only after their mass was >5 mm³ grew, although less rapidly than did tumors of the same initial size treated with LXSN alone or only with media. The tumors of all mice injected with LXSN vector alone or with media alone grew steadily, although at variable rates. Histopathologic analysis of the injected tumors did not indicate any obvious change in differentiation of tumor cells nor any induction of necrosis, indicating that LXSN-BRCA1 suppresses tumors by inhibition of growth.

In summary, the human prostate tumors injected with the LXSN-BRCA1 retroviruses were 20 fold smaller than control tumors by 26 days after viral injection (uninjected control tumors, 109 (62 mm³, n=6); LXSN tumors, 89 (32 mm³, n=11); and LXSN-BRCA1 tumors, 5.4 (2 mm³, n=16) (Table 2). Moreover, 9/16 tumors in the LXSN-BRCA1 group completely disappeared by 26 days (Table 2). BRCA1 replacement by retroviral gene therapy dramatically suppresses human prostate cancer in the nude mouse model.

TABLE 2 Growth (mass in mm³) of PPC-1 tumors in mice after injection of retroviral BRCA1, retrovirus alone, or control Day 0 Day 16 Day 26 Initial tumors <2 mm³ LXSN-BRCA1 0.52 0 0 LXSN-BRCA1 0.52 0 0 LXSN-BRCA1 0.52 0 0 LXSN-BRCA1 0.52 0 0 LXSN-BRCA1 0.52 0.98 0 LXSN-BRCA1 0.98 0 0 LXSN-BRCA1 1.57 0 0 LXSN 0.52 4.20 14.10 LXSN 1.77 14.10 14.40 Control 0.52 5.89 21.99 Initial tumors 2.7-4.2 mm³ LXSN-BRCA1 4.20 0 0 LXSN-BRCA1 4.20 0 0 LXSN-BRCA1 1.20 1.60 1.60 LXSN-BRCA1 1.20 7.85 22.80 LXSN-BRCA1 2.74 4.20 22.00 LXSN 2.74 17.86 22.00 LXSN 2.75 33.50 88.40 LXSN 2.75 33.50 219.90 LXSN 3.90 40.05 40.10 LXSN 4.10 33.50 110.00 LXSN 4.20 14.10 22.00 LXSN 4.20 33.50 40.10 LXSN 4.20 55.00 377.00 Control 2.74 17.67 33.50 Control 2.74 17.87 33.50 Control 2.74 22.00 86.40 Initial tumors >5 mm³ LXSN-BRCA1 5.89 4.20 7.85 LXSN-BRCA1 7.85 0.52 4.20 LXSN-BRCA1 7.85 5.90 14.10 LXSN-BRCA1 14.10 7.85 14.0 LXSN 7.85 12.80 33.50 Control 5.89 17.87 33.50 Control 7.85 86.40 447.60

Overexpression of wild-type BRCA1 inhibits the growth of some prostate cancer cells but does not affect growth of other prostate cancer cell lines. Near full-length truncated BRCA1 proteins do not inhibit prostate cancer cell lines, showing similarities to breast cancer but not ovarian cancer phenotype. The variable BRCA1 expression and heterogeneous response to BRCA1 transfection suggest that BRCA1 contributes to prostate cancer pathogenesis in a complex manner.

Prostate cancer cell lines appear to show loss of heterozygosity (LOH) at chromosome 17 with some frequency. However, BRCA1 mRNA and protein levels do not clearly correlate with (LOH), or with androgen receptor status. This suggests a complex relationship between somatic allele loss of chromosome 17 and the expression level of BRCA1. Thus, until the disclosure of the instant application, a therapeutic method for prostate cancer treatment using the BRCA1 gene has not been suggested.

Gene transfer of wild-type BRCA1 into prostate cancer cell lines produced inhibition in some cell lines but no inhibition in other lines. This contrasts the results obtained following transfection of wild-type BRCA1 into breast and ovarian cancer cells which are generally inhibited, although some breast and ovarian cancer cell lines are not inhibited. The ability of transfected BRCA1 to inhibit prostate cancer cell growth did not cleanly correlate with (LOH), expression level or androgen receptor status although larger number of cell lines must be studied before these potential correlations can be completely excluded. Cells which were inhibited by wild-type BRCA1 transfection were not inhibited by transfection of truncation mutants or missense mutants (Table 1). The C-terminal mutant 1835stop did not inhibit the growth of prostate cancer cells. This mutant has previously been shown to inhibit the growth of breast cancer cells but not ovarian cancer cells, suggesting that the mechanism of inhibition of prostate cancer cells by BRCA1 shows similarities to inhibition of breast cancer but not ovarian cancer.

The mechanism of PPC-1 tumor suppression by LXSN-BRCA1 may be explained on the basis of growth inhibition since LXSN-BRCA1 growth inhibits PPC-1 cells in in vitro tissue culture studies.

Tumor suppression by LXSN-BRCA1 was dependent on tumor size (Table 2). This data is most consistent with a gene-based tumor suppression and not an immune-based gene therapy which might produce a generalized effect. Previous experience with this injection protocol indicates that this experimental approach results in retroviral vector integration into 20 to 40% of tumor cells adjacent to the site of injection. These results suggest that direct injection of retroviral vectors is more effective for tumors less than 1 cm³. However, a 4.2 cm³ tumor was eliminated by this approach. Repeated or multiple injections should allow effective treatment of larger tumors, as has been demonstrated in other model systems.

These results taken together suggest that BRCA1 contributes to pathogenesis of prostate cancer in a more phenotypically complex manner than breast or ovarian cancer. There is much more heterogeneity in results obtained with prostate cancer cells than was observed with analysis of breast and ovarian cancer. Whereas most breast and ovarian cancer lines show low expression of BRCA1, prostate cancer cell lines show variable expression. Similarly, the observation that transfection of BRCA1 inhibits only a proportion of prostate cancer cells emphasizes the heterogeneity of prostate cancer and suggests that prostate cancer cells may differ in BRCA1 signaling. This may explain why BRCA1 mutation produces only a relatively small increased risk of prostate cancer.

EXAMPLE 3 LXSN-BRCA1 Retroviral Therapy of Advanced Prostate Cancer

This example describes novel corrective prostate specific viral based gene therapy to combat advanced prostate cancer. Corrective gene therapy attempts to correct genetic mutations in cancer by replacing mutated tumor suppressor genes with normal ones. LXSN:BRCA1 retroviral gene therapy is applied to advanced prostate cancer by in vivo gene transfer of BRCA1 sequences with expression regulated by the Moloney long terminal repeat (LTR). Preclinical studies have revealed that prostate cancer cells that have low expression of BRCA1 protein are inhibited by the transfection of wild type BRCA1 and that small human prostate tumors established in nude mice are inhibited by the injection of a retroviral vector expressing wild type BRCA1. Transduction with these viral vectors results in marked tumor inhibition or even cure of some experimental animals with no clear-cut toxicity. The tissue selectivity of inhibition by BRCA1 may contribute to the limited toxicity which we have observed in studies in nude mice. Therefore, this example describes application of this method for the treatment of human advanced prostate cancer.

This example focuses on maximizing the delivery of retroviral vector to the tumor cells by repeated administrations into the orthotopic cancerous prostate in an attempt to increase the antitumor effect. Patients undergo a tissue examination prior to injection of retroviral vector (transrectal ultrasound quadrant injections). Pathologic, biochemical, and molecular studies are performed on biopsies to follow the extent of viral vector uptake by tumor cells and determine the stability of the viral vector. The clinical extent of tumor spread is measured before and after retroviral vector injection by clinical exam, ultrasound measurement of tumor volume, and serum prostate specific antigen (PSA).

Under transrectal ultrasound guidance, four needle cores of cells are removed (one from each prostate quadrant) per session and examined by methods cited above. Then, the retroviral vector is injected into the space left by the biopsy. The initial studies and injections are performed as an in-patient procedure within the Clinical Research Center, University of Tennessee-Memphis. Following the fourth injection session, the patient is discharged and then returns at two weeks and at four weeks for follow-up. In the event of death, a post-mortem examination quantifies tumor spread by careful dissection, measurement of tumor volume and weight, microscopically directed analysis of tumor extent, and molecular analysis of tumor and adjacent normal tissues to compare the extent of gene transfer between tumor cells and adjacent normal cells. More extensive tumor seeding requires repeated treatments with retroviral vector in order to achieve a therapeutic response, particularly since large tumors may be composed predominantly of slowly dividing cells which may require repeated exposure of the tumor cells to retroviral vector.

Overview of Therapy

Patients with advanced prostate cancer who meet the study criteria are treated with retroviral gene therapy by injection of retroviral vectors into the orthotopic prostate tumor. Retroviral vectors are manufactured from viral producer cells using serum-free conditions and are tested for sterility, absence of specific pathogens, and an absence of replication-competent retrovirus by standard assays. Retrovirus are stored frozen in large aliquots which have been tested according to FDA standards.

Patients are admitted to the Clinical Research Center, University of Tennessee-Memphis where they have a complete physical exam, blood, and urine tests to determine overall health. They bring with them a current bone scan, chest X-ray, electrocardiogram, and appropriate radiologic procedures to assess tumor stage.

Patients spend four days in the Clinical Research Center, University of Tennessee-Memphis for the initial injections of retroviral vector. Blood samples are drawn each day and tested for the presence of retroviral vector by sensitive polymerase chain reaction (PCR)-based assays. Patients with advanced prostate cancer have the cancer cells from their initial prostate biopsy analyzed to determine:

1. The percentage of cancer cells which are taking up the vector/gene combination by PCR and by in-situ hybridization;

2. The number of cancer cells present in the biopsy (cancer cell density);

3. Differentiation status of the cells (alcian blue/PAS);

4. Presence of programmed cell death (ApoTAG and DNA analysis);

5. Measurement of expression of BRCA1 target gene by immunohistochemistry and Western blot analysis.

Patients are continuously monitored while in the Clinical Research Center, University of Tennessee-Memphis. After the four day period in the Clinical Research Center they are discharged. Depending upon clinical status, they are either discharged to the Urology Division or to home, but all patients are asked to return at day 7 for a blood sample. After 4 weeks from the completion of the virus vector injections, the patients are reevaluated and undergo a prostate biopsy. After this evaluation the patients then proceed with chemotherapy or other options as clinically indicated to control temporarily their disease. Table 3 summarizes preliminary evaluation, screening, and treatment evaluation.

Maximally tolerated dose (MTD) of LXSN-BRCA1 when administered directly into the cancerous prostate is determined. Primary endpoints are: 1) the rate of transduction in tumor and/or normal cells, 2) the presence and stability of this vector in the systemic circulation and in prostate cancer cells, and 3) the nature of the systemic (fever, myalgias) and local (infections, pain) toxicities induced by this vector. A secondary endpoint is the clinical efficacy of LXSN-BRCA1.

Eligible patients with advanced prostate cancer are admitted to the Clinical Research Center, University of Tennessee-Memphis (CRC). Inclusion criteria are as follows:

1. Advanced prostate cancer

2. Patients who are >35 and <75 years old and who have signed informed consent

3. ECOG performance status (PS) ≧2

4. Life expectancy of greater than 6 months

5. Recovery for at least 4 weeks from previous surgery and/or other cancer therapies

6. Adequate hematological (WBC's >4,000/mm³, platelet count >100,000/mm³), hepatic (bilirubin <mg/dL, SGOT <2×normal), and renal (creatinine <1.5 mg/dl) functions.

Exclusion criteria are as follows:

1. Localized prostate cancer

2. Active bacterial infections

3. Patients on concomitant experimental or other alternative therapies

4. Patients with heart failure (NYHA class 4), recent myocardial infarction, respiratory insufficiency, or hematological, hepatic, or renal dysfunction

5. Concomitant anticoagulant or antiplatelet drugs

6. Previous radiotherapy.

Selection is also based on presence of measurable disease, ECOG score, and inclusion/exclusion criteria set forth above. Patients are recruited through contacts with urologists, medical oncologists, and radiation oncologists who are currently providing care to the patient. Individual discussions with the patient and family members are scheduled to answer all concerns and questions about the method.

Prostate cancer tissue is collected for molecular studies by transrectal ultrasound guided biopsy using a biopsy gun. The vector was produced under current Good Manufacturing Practices and is provided by the Vector Production Facility at Vanderbilt University.

A 4 ml serum-free volume of retroviral vector (containing up to 5×10⁷ viral particles in AIM V media) is administered daily per session. During each session, 1 ml of medium containing the appropriate titer of LXSN-BRCA1 is injected under transrectal ultrasound guidance into 4 regions of the prostate for a total of 4 ml per session in a clinical examination room. This is repeated daily for 4 days (4 sessions). Since the rectal wall is insensate, the patient should experience very little discomfort. This 16 ml total inoculum volume over 4 days is proportionally well below the one safely tolerated by nude mice (0.5 ml/20 g body weight). Moreover, the biopsy of 16 different areas of the prostate by transrectal ultrasound guidance assures representative sampling of the prostate.

Patient evaluation includes history and physical examination prior to initiation of therapy and daily during the 4 day period of vector injection. Toxicity grading is done using the ECOG Common Toxicity Criteria. CBC, SMA-20, urinalysis, and conventional studies are performed daily during this period (see Table 3 which presents parameters). Patients are allowed to proceed with any standard palliative alternatives (i.e., systemic chemotherapy) after the completion of vector administration. However, it is not expected that all patients will require immediate additional palliative interventions.

Dose Escalation and MTD

Three patients are treated with 3×10⁶ viral particles×4. Once they have all recovered from all grade 2 or less toxicities (except alopecia), and as long as grade 3-4 toxicity is not encountered, a subsequent dose level is initiated in 3 additional patients. As one grade 3 or 4 toxicity occurs at a given dose level, a minimum of 6 patients are enrolled at that level. As only 1 of 6 patients has grade 3 or 4 toxicity, dose escalation continues. The MTD of LXSN-BRCA1 is defined as the dose where 2 of 6' patients experience grade 3 or 4 toxicity. If 2 of 3, or if 3 of 6 patients experience grade 3 or 4 toxicity, the MTD is defined as the immediately lower dose level.

The following escalation schema is followed: 1) level 1, 3×10⁶ viral particles; 2) level 2, 1×10⁷; 3) level 3, 3×10⁷; 4) level 4, 5×10⁷.

Studies of Retroviremia

Previous preclinical data indicate that injection of relatively large quantities of vector into the peritoneal space results in detectable amounts of vector in the peripheral blood in mice, although detectable vector in peripheral blood in patients treated with up to 10¹⁰ vector transducing units has not been observed. This problem may be explained by the large volume of literature which indicates that human serum destroys retroviral particles. This issue is addressed in patients by obtaining 20 ml of blood during each of the four days that the patients are present in the Clinical Research Center, University of Tennessee-Memphis, and separating the blood into serum and cellular components for PCR detection.

If the viral vector is detected within the serum component, then the following assay to identify the existence of transduction-capable viral vector is performed. Serum is incubated with PPC-1 target cells, and DNA is obtained from PPC-1 cells before and after attempted transduction.

Criteria for Clinical Response

Patients with measurable disease are evaluated for a clinical response to LXSN-BRCA1, especially those that do not undergo a palliative intervention immediately after retroviral vector therapy. Prostate histology, prostatic volume by ultrasound, PSA, and local symptoms are followed. For other sites of disease, conventional response criteria are used as follows:

1. Complete Response (CR)—complete disappearance of all measurable lesions and of all signs and symptoms of disease for at least 4 weeks.

2. Partial Response (PR)—decrease of at least 50% of the sum of the products of the 2 largest perpendicular diameters of all measurable lesions as determined by 2 observations not less than 4 weeks apart. To be considered a PR, no new lesions should have appeared during this period and none should have increased in size.

3. Stable Disease—less than 25% change in tumor volume from previous evaluations.

4. Progressive Disease—greater than 25% increase in tumor measurements from previous evaluations.

Potential Risks

1. Blood collection

Bruising and infection.

2. Prostate biopsy

There will be some discomfort and the possibility of bleeding or infection related to the biopsy. In rare instances, this infection can lead to fever.

3. Vector injection—It is possible that a person may have an allergic reaction to the injection although this should be a rare complication since no animal serum is used to prepare the vector for injection. The retroviral vector may kill tumor cells producing necrosis and release of these factors into the blood stream resulting in fever, changes in blood chemistry, changes in white blood cell count, and the possibility of uric acid kidney stones.

4. Retroviral vector replication—The retroviral vectors employed herein are unable to reproduce or replicate. Unknown or uncommon side effects may occur including ones that may be severe since this is a new form of cancer treatment.

5. Safety precautions

1) Blood collection: The site is swabbed with alcohol to minimize the risk of infection. In addition, pressure is placed in the venipuncture site to prevent bruising or bleeding.

2) Prostate biopsy: The patient receives antibiotic prophylaxis with a Cipro 500 mg PO BID the day before biopsy, the day of biopsy, and the day after biopsy. This has been proven to be effective in preventing infection related to a transrectal biopsy. Pressure can be maintained at the site of biopsy to minimize bleeding.

3) Vector injection: As mentioned above, the vector is prepared as specified by the FDA including the use of AIM V which is an animal serum-free media. The packaging cell lines have been fully tested free of any other potential pathogens.

4) Vector replication: Blood samples and tissue are tested on a routine basis for the presence of helper virus activity. The patients are followed very closely to see if any side effects are indeed occurring which is the reason that they spend 4 full days in the CRC during treatment.

All data is collected and tabulated with the utmost concern for the patient's privacy and confidentiality. The data includes molecular studies of blood and prostate tissue in addition to history and physical examination, tumor status, performance status, toxicity assessments, weight, complete blood counts, PT/PTT, urinalysis, blood urea nitrogen & creatinine, liver function tests, serum chemistries, chest x-ray, electrocardiogram, and serum prostate-specific antigen. Prostate tissue, fixed or frozen, as well as serum samples are stored by number in a −70° freezer.

TABLE 3 Study Parameters for Clinical Trial Flow Sheet PreTreat- 4 ment 2 weeks weeks Routine Daily during post- Monthly Studies (Rx)⁵ Rx RX post-Rx X11 Yearly History & X X X X X X Physical Assess X X X X X X Tumors Status Perform- X X X X X X ance Status Toxicity X X X X X X Assess- ment Weight X X X X X X X Complete X X X X X X Blood Count PT,PTT X X X X X X Urin- X X X X X X alysis BUN & X X X X X X Creati- nine Liver X X X X X X Function Tests² Serum X X X X X X Chem- istries³ Chest X ACI ACI ACI ACI ACI X-ray EKG X ACI ACI ACI ACI ACI PSA X X X X X X Circulat- X X X X X X ing env anti- bodies Prostate X X X X biopsy ¹To include: hematocrit, hemoglobin, differential, and platelets ²To include: alkaline phosphatase, serum transaminases, bilirubin, protein, LDH, and albumin ³To include: Na, K, Ca, PO4, Cl, Magnesium, CO2, and glucose

Table 4 presents the partial response observed in six out of nineteen patients who have been treated with the LXSN-BRCA1 gene therapy methods described herein. As more fully defined above, a partial response is defined as greater than 50% tumor shrinkage. The data indicate the percent tumor size shrinkage observed in these patients. This is determined by doing ultrasounds on the tumors before and after therapy.

TABLE 4 Patient Number Percent Tumor Size Shrinkage 1 53 2 50 3 51 4 54 5 61 6 52

EXAMPLE 4 Gene Therapy of Prostate Cancer Using the BRCA2 Gene.

The protein encoded by the BRCA2 breast and ovarian cancer susceptibility gene (Wooster, R. et al. 1995) includes a domain similar to the granin consensus at the C-terminus of the protein. As seen in FIG. 5, the sequence at amino acids 3334-3344 of Genbank locus HUS43746 matches six of the seven constrained sites of the granin consensus. BRCA2 and murine BRCA1 differ from the consensus at the same site. The granin motif in BRCA2 lies at the extreme C-terminal end of the protein, a locale characteristic of a known granin. This indicates that the protein encoded by the BRCA2 gene is also a secreted growth inhibitor. Use of both the BRCA1 and BRCA2 genes offers the opportunity for a unified approach to the treatment of prostate cancer. Accordingly, the examples set forth above depicting the treatment of prostate cancer, are equally applicable to the BRCA2 gene and the BRCA2 gene product.

The identification of BRCA1 and BRCA2 as granins indicate that there is a granin superfamily which consists of the subfamilies of chromogranins (chromogranins A, B and C); secretogranins (secretogranins III-V) and the BRCAgranins (BRCA1, BRCA2 and other tumor suppressor genes). This classification of granin into these subclasses is based on greater similarities within the subfamilies than with the superfamily as a whole. For example, chromogranins share an additional region of homology besides the granin consensus and exhibit similar expression patterns; the secretogranins show less homology to the granin consensus than either chromogranins or BRCA granins; the BRCA granins BRCA1 and BRCA2 are cancer susceptibility genes, contain additional regions of homology, and are significantly larger (two-twenty times larger) than other granins described to date.

Thus, the invention provides in Example 3 and in this Example a granin box consensus sequence shown in FIG. 5. Thus, provided is a family of proteins which share the consensus sequence and that are tumor suppressor genes. BRCA1 and BRCA2 are members of this family. Other members may be identified and purified as tumor suppressor genes by genetic methods, by DNA-based searches for granin homology, or by cloning and characterization of granins in prostate cancer cells by biochemical methods. Such biochemical methods include the isolation and purification of proteins from the secretory vesicles or Golgi by physical isolation methods, followed by development of antibodies to determine which proteins, followed by cloning of genes for secreted proteins after protein sequencing and cloning with degenerate oligonucleotide primers. An example of this method is described in Colomer et al., 1996. Thus, other BRCA granins are contemplated to be within the scope of this invention. Accordingly, the therapy methods described herein are contemplated to be effective using other BRCA granins as well as BRCA1 and BRCA2.

Therefore, the term “BRCA family” as used herein and in the claims, is contemplated to include the BRCA granins described in this Example as well as BRCA1 and BRCA2 genes and gene products. The BRCA family is characterized by the tumor suppressor activity of the gene product and the granin box consensus sequence shown in FIG. 5.

EXAMPLE 5 Gene Transfer Using Liposomes

An alternative method of gene therapy using the BRCA1 and BRCA2 gene, and BRCA gene family includes the use of liposomes to deliver the DNA into the cells. By this method, the above described LXSN-BRCA1 plasmid is incubated with a liposome preparation such as cationic liposomes and then the DNA liposome mix is added to cells or injected into an animal or patient. Generally, the liposome transfection method is of a lower efficiency than viral gene transfer methods. This method is made more useful because the BRCA1 and BRCA2 and BRCA granin proteins are secreted proteins. Thus, if only a few percent of cells take up the DNA-liposome combination, it is likely that enough gene product will be produced and secreted from these cells to growth inhibit other cells. Liposomal transfection of nucleic acids into host cells is described in U.S. Pat. Nos. 5,279,833; 5,286,634; 5,651,964; 5,641,484; and 5,643,567, the contents of each of which are herein incorporated by reference.

EXAMPLE 6 Anti-sense Inhibition of the Production of BRCA1 Protein

The antisense inhibition of BRCA1 is described as follows. Antisense methods are used to demonstrate that BRCA1 expression inhibits cell growth. Unmodified 18 base deoxyribonucleotide complementary to the BRCA1 translation initiation site are synthesized and are added to cultures of primary prostate cancer cells at a concentration of 40 μM according to well-known procedures.

Upon acceleration of the growth of prostate cancer cells via antisense inhibition of BRCA1, chemotherapeutic methods of treating prostate cancer are improved. Because chemotherapy is most effective in cancer cells which are rapidly dividing, it is possible then to treat prostate cancer by accelerating growth of cancer cells by antisense inhibition of BRCA1 protein expression and by treating with chemotherapeutic drugs using standard chemotherapy protocols.

EXAMPLE 7 Treatment of Prostate Cancer Using Purified BRCA1 or BRCA2 Gene Product

Alternatively, prostate cancer is treated by the administration of a therapeutically effective amount of the BRCA1 or BRCA2 gene product via an efficient method, such as injection into a tumor. A therapeutically effective amount can be determined by one having ordinary skill in the art using well-known protocols.

It is important to note that prostate cancer cells have surface receptors which can be contacted by the BRCA1 or BRCA2 gene product. Thus, the BRCA1 or BRCA2 gene product, an active fragment, or a small molecule mimetic binds directly to a receptor on the surface of the prostate cancer cells. BRCA1 and BRCA2 targeted growth inhibitor agents as defined herein are preferred examples.

EXAMPLE 8 Method of Treating Prostate Cancer Comprising Introducing the BRCA1 Receptor Gene and the BRCA1 Protein into a Prostate Cancer Cell

The loss of the BRCA1 receptor in prostate cancer cells will lead to proliferation and tumorigenesis in these cells. Thus, prostate cancer can be treated by introducing the BRCA1 receptor gene into prostate cancer cells using the gene therapy methods described above. This step will be followed by the administration of a therapeutically effective amount of the BRCA1 gene product so that the BRCA1 gene product contacts a receptor on a surface of the prostate cells. A therapeutically effective amount can be determined by one having ordinary skill in the art using well-known protocols.

The BRCA1 receptor gene is isolated using standard techniques. The BRCA2 receptor gene can be similarly isolated.

Baculovirus BRCA1 is purified from the insect cells with an antibody derived from the last twenty amino acids of the carboxy terminius of the BRCA1 gene product (the C20 antibody) and then labeled with radioactive iodine by standard methods. Cys61Gly and termination codon mutant BRCA1 proteins are prepared and labelled as a control. The labelled BRCA1 then can be used to perform binding studies to identify cells with BRCA1 receptors using Scatchard analysis and to perform cross-linking studies which demonstrate the BRCA1 receptor(s) on polyacrylamide gels. These initial characterization methods are used to identify cells with high and low numbers of BRCA1 receptor(s) for purification and isolation studies. Once a cell line with high levels of BRCA1 receptor has been identified, then the protein is purified by the following approaches:

Approach A: Biochemical purification.

The cell line which expresses high levels of BRCA1 receptor is lysed and the protein from cell lysates or membrane preparations is purified by gel filtration, followed by purification of the receptor with a column containing the BRCA1 ligand bound to a solid phase such as sepharose. The purified receptor protein can then be microsequenced and the gene cloned using degenerate oligonucleotides derived from the protein sequence.

Approach B:

Ligand is radiolabeled with 125I and then used to screen cell lines or tissues for specific binding by Scatchard analysis. Once such binding is identified, a cDNA library is constructed from that tissue or cell line and transfected into a cell line that does not exhibit specific binding. These transfected cells are then screened for newly acquired specific binding which indicates they have been transfected with a construct containing the gene for the BRCA1 receptor. Plasmid DNA from positive clones is then isolated and sequenced for identification. This single construct is then transfected back into the null cells to verify that binding of ligand is mediated by the transfected gene. (Kluzen et al. 1992).

Alternatively, chimeric BRCA1 and immunoglobulin Fc molecules can be constructed. (LaRochelle et al. 1995). The chimeric molecules are then used to screen for binding to BRCA1 receptor on whole cells via flow cytometry. Alternatively, due to the presence of the immunoglobulin component of the molecule, cell lysates are screened by immunoblotting or by immunoprecipitation of metabolically labelled cells. This technique can identify BRCA1 binding proteins by a variety of different methods. Peptide digests of the identified proteins are then generated so that the peptides can be sequenced and the whole molecule cloned by a degenerative oligonucleotide approach.

EXAMPLE 9 Method of Preventing Prostate Cancer Using BRCA1 or BRCA2 Protein

BRCA1 gene product is used as a chemopreventive agent by introducing BRCA1 directly into the prostate as the whole protein, as a functional fragment, or as a functional cleavage product. In addition, compounds that induce expression of BRCAL or activate its receptor, e.g., a small molecule mimetic, could also be introduced.

Gene therapy approaches for increasing the expression of BRCA1 in the prostate gland directly or indirectly could also be used. Systemic agents that induce the expression of BRCA1, or that mimic function and can replace BRCA1, such a peptidomimetic agent, could also be used. The delivery of such agents could take place by directly instilling the agent within the prostate. Finally, an implantable time release capsule can be used in a prevention strategy, by placing such a capsule in the prostate for prostate cancer.

Since the BRCA2 protein includes a granin sequences and is also a secreted tumor suppressor protein, similar prevention strategies can be applied using the BRCA2 gene and protein.

Thus, because patients with mutations in BRCA1 or BRCA2 have an increased incidence of prostate cancer, overexpression of BRCA1 or BRCA2 genes (or stimulated expression of endogenous BRCA1 or BRCA2 genes) is likely useful in preventing the development of prostate cancer.

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It will be understood that various details of the invention may be changed without departing from the scope of the invention. Furthermore, the foregoing description is for the purpose of illustration only, and not for the purpose of limitation—the invention being defined by the claims.

                   #             SEQUENCE LISTING (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 26 (2) INFORMATION FOR SEQ ID NO:1:      (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 5712           (B) TYPE: nucleic acid           (C) STRANDEDNESS: double           (D) TOPOLOGY: linear     (ix) FEATURE:           (A) NAME/KEY: BRCA1           (B) LOCATION: GenBank a #ccession no. U14680      (x) PUBLICATION INFORMATION:           (A) AUTHORS:  Miki,  #Y., et. al.           (B) TITLE: A strong  #candidate gene for the breast and                ovarian c #ancer susceptibility gene BRCA1.           (C) JOURNAL: Science           (D) VOLUME: 266           (E) PAGES: 66-71           (F) DATE: 1994     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: #1: agctcgctga gacttcctgg accccgcacc aggctgtggg gtttctcaga ta #actgggcc     60 cctgcgctca ggaggccttc accctctgct ctgggtaaag ttcattggaa ca #gaaagaa     119 atg gat tta tct gct ctt cgc gtt gaa gaa gt #a caa aat gtc att aat      167 Met Asp Leu Ser Ala Leu Arg Val Glu Glu Va #l Gln Asn Val Ile Asn 1               5    #                10   #                15 gct atg cag aaa atc tta gag tgt ccc atc tg #t ctg gag ttg atc aag      215 Ala Met Gln Lys Ile Leu Glu Cys Pro Ile Cy #s Leu Glu Leu Ile Lys             20       #            25       #            30 gaa cct gtc tcc aca aag tgt gac cac ata tt #t tgc aaa ttt tgc atg      263 Glu Pro Val Ser Thr Lys Cys Asp His Ile Ph #e Cys Lys Phe Cys Met         35           #        40           #        45 ctg aaa ctt ctc aac cag aag aaa ggg cct tc #a cag tgt cct tta tgt      311 Leu Lys Leu Leu Asn Gln Lys Lys Gly Pro Se #r Gln Cys Pro Leu Cys     50               #    55               #    60 aag aat gat ata acc aaa agg agc cta caa ga #a agt acg aga ttt agt      359 Lys Asn Asp Ile Thr Lys Arg Ser Leu Gln Gl #u Ser Thr Arg Phe Ser 65                   #70                   #75                   #80 caa ctt gtt gaa gag cta ttg aaa atc att tg #t gct ttt cag ctt gac      407 Gln Leu Val Glu Glu Leu Leu Lys Ile Ile Cy #s Ala Phe Gln Leu Asp                 85   #                90   #                95 aca ggt ttg gag tat gca aac agc tat aat tt #t gca aaa aag gaa aat      455 Thr Gly Leu Glu Tyr Ala Asn Ser Tyr Asn Ph #e Ala Lys Lys Glu Asn             100       #           105       #           110 aac tct cct gaa cat cta aaa gat gaa gtt tc #t atc atc caa agt atg      503 Asn Ser Pro Glu His Leu Lys Asp Glu Val Se #r Ile Ile Gln Ser Met         115           #       120           #       125 ggc tac aga aac cgt gcc aaa aga ctt cta ca #g agt gaa ccc gaa aat      551 Gly Tyr Arg Asn Arg Ala Lys Arg Leu Leu Gl #n Ser Glu Pro Glu Asn     130               #   135               #   140 cct tcc ttg cag gaa acc agt ctc agt gtc ca #a ctc tct aac ctt gga      599 Pro Ser Leu Gln Glu Thr Ser Leu Ser Val Gl #n Leu Ser Asn Leu Gly 145                 1 #50                 1 #55                 1 #60 act gtg aga act ctg agg aca aag cag cgg at #a caa cct caa aag acg      647 Thr Val Arg Thr Leu Arg Thr Lys Gln Arg Il #e Gln Pro Gln Lys Thr                 165   #               170   #               175 tct gtc tac att gaa ttg gga tct gat tct tc #t gaa gat acc gtt aat      695 Ser Val Tyr Ile Glu Leu Gly Ser Asp Ser Se #r Glu Asp Thr Val Asn             180       #           185       #           190 aag gca act tat tgc agt gtg gga gat caa ga #a ttg tta caa atc acc      743 Lys Ala Thr Tyr Cys Ser Val Gly Asp Gln Gl #u Leu Leu Gln Ile Thr         195           #       200           #       205 cct caa gga acc agg gat gaa atc agt ttg ga #t tct gca aaa aag gct      791 Pro Gln Gly Thr Arg Asp Glu Ile Ser Leu As #p Ser Ala Lys Lys Ala     210               #   215               #   220 gct tgt gaa ttt tct gag acg gat gta aca aa #t act gaa cat cat caa      839 Ala Cys Glu Phe Ser Glu Thr Asp Val Thr As #n Thr Glu His His Gln 225                 2 #30                 2 #35                 2 #40 ccc agt aat aat gat ttg aac acc act gag aa #g cgt gca gct gag agg      887 Pro Ser Asn Asn Asp Leu Asn Thr Thr Glu Ly #s Arg Ala Ala Glu Arg                 245   #               250   #               255 cat cca gaa aag tat cag ggt agt tct gtt tc #a aac ttg cat gtg gag      935 His Pro Glu Lys Tyr Gln Gly Ser Ser Val Se #r Asn Leu His Val Glu             260       #           265       #           270 cca tgt ggc aca aat act cat gcc agc tca tt #a cag cat gag aac agc      983 Pro Cys Gly Thr Asn Thr His Ala Ser Ser Le #u Gln His Glu Asn Ser         275           #       280           #       285 agt tta tta ctc act aaa gac aga atg aat gt #a gaa aag gct gaa ttc     1031 Ser Leu Leu Leu Thr Lys Asp Arg Met Asn Va #l Glu Lys Ala Glu Phe     290               #   295               #   300 tgt aat aaa agc aaa cag cct ggc tta gca ag #g agc caa cat aac aga     1079 Cys Asn Lys Ser Lys Gln Pro Gly Leu Ala Ar #g Ser Gln His Asn Arg 305                 3 #10                 3 #15                 3 #20 tgg gct gga agt aag gaa aca tgt aat gat ag #g cgg act ccc agc aca     1127 Trp Ala Gly Ser Lys Glu Thr Cys Asn Asp Ar #g Arg Thr Pro Ser Thr                 325   #               330   #               335 gaa aaa aag gta gat ctg aat gct gat ccc ct #g tgt gag aga aaa gaa     1175 Glu Lys Lys Val Asp Leu Asn Ala Asp Pro Le #u Cys Glu Arg Lys Glu             340       #           345       #           350 tgg aat aag cag aaa ctg cca tgc tca gag aa #t cct aga gat act gaa     1223 Trp Asn Lys Gln Lys Leu Pro Cys Ser Glu As #n Pro Arg Asp Thr Glu         355           #       360           #       365 gat gtt cct tgg ata aca cta aat agc agc at #t cag aaa gtt aat gag     1271 Asp Val Pro Trp Ile Thr Leu Asn Ser Ser Il #e Gln Lys Val Asn Glu     370               #   375               #   380 tgg ttt tcc aga agt gat gaa ctg tta ggt tc #t gat gac tca cat gat     1319 Trp Phe Ser Arg Ser Asp Glu Leu Leu Gly Se #r Asp Asp Ser His Asp 385                 3 #90                 3 #95                 4 #00 ggg gag tct gaa tca aat gcc aaa gta gct ga #t gta ttg gac gtt cta     1367 Gly Glu Ser Glu Ser Asn Ala Lys Val Ala As #p Val Leu Asp Val Leu                 405   #               410   #               415 aat gag gta gat gaa tat tct ggt tct tca ga #g aaa ata gac tta ctg     1415 Asn Glu Val Asp Glu Tyr Ser Gly Ser Ser Gl #u Lys Ile Asp Leu Leu             420       #           425       #           430 gcc agt gat cct cat gag gct tta ata tgt aa #a agt gaa aga gtt cac     1463 Ala Ser Asp Pro His Glu Ala Leu Ile Cys Ly #s Ser Asp Arg Val His         435           #       440           #       445 tcc aaa tca gta gag agt aat att gaa gac aa #a ata ttt ggg aaa acc     1511 Ser Lys Ser Val Glu Ser Asp Ile Glu Asp Ly #s Ile Phe Gly Lys Thr     450               #   455               #   460 tat cgg aag aag gca agc ctc ccc aac tta ag #c cat gta act gaa aat     1559 Tyr Arg Lys Lys Ala Ser Leu Pro Asn Leu Se #r His Val Thr Glu Asn 465                 4 #70                 4 #75                 4 #80 cta att ata gga gca ttt gtt act gag cca ca #g ata ata caa gag cgt     1607 Leu Ile Ile Gly Ala Phe Val Ser Glu Pro Gl #n Ile Ile Gln Glu Arg                 485   #               490   #               495 ccc ctc aca aat aaa tta aag cgt aaa agg ag #a cct aca tca ggc ctt     1655 Pro Leu Thr Asn Lys Leu Lys Arg Lys Arg Ar #g Pro Thr Ser Gly Leu             500       #           505       #           510 cat cct gag gat ttt atc aag aaa gca gat tt #g gca gtt caa aag act     1703 His Pro Glu Asp Phe Ile Lys Lys Ala Asp Le #u Ala Val Gln Lys Thr         515           #       520           #       525 cct gaa atg ata aat cag gga act aac caa ac #g gag cag aat ggt caa     1751 Pro Glu Met Ile Asn Gln Gly Thr Asn Gln Th #r Glu Gln Asn Gly Gln     530               #   535               #   540 gtg atg aat att act aat agt ggt cat gag aa #t aaa aca aaa ggt gat     1799 Val Met Asn Ile Thr Asn Ser Gly His Glu As #n Lys Thr Lys Gly Asp 545                 5 #50                 5 #55                 5 #60 tct att cag aat gag aaa aat cct aac cca at #a gaa tca ctc gaa aaa     1847 Ser Ile Gln Asn Glu Lys Asn Pro Asn Pro Il #e Glu Ser Leu Glu Lys                 565   #               570   #               575 gaa tct gct ttc aaa acg aaa gct gaa cct at #a agc agc agt ata agc     1895 Glu Ser Ala Phe Lys Thr Lys Ala Glu Pro Il #e Ser Ser Ser Ile Ser             580       #           585       #           590 aat atg gaa ctc gaa tta aat atc cac aat tc #a aaa gca cct aaa aag     1943 Asn Glu Leu Glu Leu Asn Ile Met His Asn Se #r Lys Ala Pro Lys Lys         595           #       600           #       605 aat agg ctg agg agg aag tct tct acc agg ca #t att cat gcg ctt gaa     1991 Asn Arg Leu Arg Arg Lys Ser Ser Thr Arg Hi #s Ile His Ala Leu Glu     610               #   615               #   620 cta gta gtc agt aga aat cta agc cca cct aa #t tgt act gaa ttg caa     2039 Leu Val Val Ser Arg Asn Leu Ser Pro Pro As #n Cys Thr Glu Leu Gln 625                 6 #30                 6 #35                 6 #40 att gat agt tgt tct agc agt gaa gag ata aa #g aaa aaa aag tac aac     2087 Ile Asp Ser Cys Ser Ser Ser Glu Glu Ile Ly #s Lys Lys Lys Tyr Asn                 645   #               650   #               655 caa atg cca gtc agg cac agc aga aac cta ca #a ctc atg gaa ggt aaa     2135 Gln Met Pro Val Arg His Ser Arg Asn Leu Gl #n Leu Met Glu Gly Lys             660       #           665       #           670 gaa cct gca act gga gcc aag aag agt aac aa #g cca aat gaa cag aca     2183 Glu Pro Ala Thr Gly Ala Lys Lys Ser Asn Ly #s Pro Asn Glu Gln Thr         675           #       680           #       685 agt aaa aga cat gac agc gat act ttc cca ga #g ctg aag tta aca aat     2231 Ser Lys Arg His Asp Ser Asp Thr Phe Pro Gl #u Leu Lys Leu Thr Asn     690               #   695               #   700 gca cct ggt tct ttt act aag tgt tca aat ac #c agt gaa ctt aaa gaa     2279 Ala Pro Gly Ser Phe Thr Lys Cys Ser Asn Th #r Ser Glu Leu Lys Glu 705                 7 #10                 7 #15                 7 #20 ttt gtc aat cct agc ctt cca aga gaa gaa aa #a gaa gag aaa cta gaa     2327 Phe Val Asn Pro Ser Leu Pro Arg Glu Glu Ly #s Glu Glu Lys Leu Glu                 725   #               730   #               735 aca gtt aaa gtg tct aat aat gct gaa gac cc #c aaa gat ctc atg tta     2375 Thr Val Lys Val Ser Asn Asn Ala Glu Asp Pr #o Lys Asp Leu Met Leu             740       #           745       #           750 agt gga gaa agg gtt ttg caa act gaa aga tc #t gta gag agt agc agt     2423 Ser Gly Glu Arg Val Leu Gln Thr Glu Arg Se #r Val Glu Ser Ser Ser         755           #       760           #       765 att tca ttg gta cct ggt act gat tat ggc ac #t cag gaa agt atc tcg     2471 Ile Ser Leu Val Pro Gly Thr Asp Tyr Gly Th #r Gln Glu Ser Ile Ser     770               #   775               #   780 tta ctg gaa gtt agc act cta ggg aag gca aa #a aca gaa cca aat aaa     2519 Leu Leu Glu Val Ser Thr Leu Gly Lys Ala Ly #s Thr Glu Pro Asn Lys 785                 7 #90                 7 #95                 8 #00 tgt gtg agt cag tgt gca gca ttt gaa aac cc #c aag gga cta att cat     2567 Cys Val Ser Gln Cys Ala Ala Phe Glu Asn Pr #o Lys Gly Leu Ile His                 805   #               810   #               815 ggt tgt tcc aaa gat aat aga aat gac aca ga #a ggc ttt aag tat cca     2615 Gly Cys Ser Lys Asp Asn Arg Asn Asp Thr Gl #u Gly Phe Lys Tyr Pro             820       #           825       #           830 ttg gga cat gaa gtt aac cac agt cgg gaa ac #a agc ata gaa atg gaa     2663 Leu Gly His Glu Val Asn His Ser Arg Glu Th #r Ser Ile Glu Met Glu         835           #       840           #       845 gaa agt gaa ctt gat gct cag tat ttg cag aa #t aca ttc aag gtt tca     2711 Glu Ser Glu Leu Asp Ala Gln Tyr Leu Gln As #n Thr Phe Lys Val Ser     850               #   855               #   860 aag cgc cag tca ttt gct ccg ttt tca aat cc #a gga aat gca gaa gag     2759 Lys Arg Gln Ser Phe Ala Pro Phe Ser Asn Pr #o Gly Asn Ala Glu Glu 865                 8 #70                 8 #75                 8 #80 gaa tgt gca aca ttc tct gcc cac tct ggg tc #c tta aag aaa caa agt     2807 Glu Cys Ala Thr Phe Ser Ala His Ser Gly Se #r Leu Lys Lys Gln Ser                 885   #               890   #               895 cca aaa gtc act ttt gaa tgt gaa caa aag ga #a gaa aat caa gga aag     2855 Pro Lys Val Thr Phe Glu Cys Glu Gln Lys Gl #u Glu Asn Gln Gly Lys             900       #           905       #           910 aat gag tct aat atc aag cct gta cag aca gt #t aat atc act gca ggc     2903 Asn Glu Ser Asn Ile Lys Pro Val Gln Thr Va #l Asn Ile Thr Ala Gly         915           #       920           #       925 ttt cct gtg gtt ggt cag aaa gat aag cca gt #t gat aat gcc aaa tgt     2951 Phe Pro Val Val Gly Gln Lys Asp Lys Pro Va #l Asp Asn Ala Lys Cys     930               #   935               #   940 agt atc aaa gga ggc tct agg ttt tgt cta tc #a tct cag ttc aga ggc     2999 Ser Ile Lys Gly Gly Ser Arg Phe Cys Leu Se #r Ser Gln Phe Arg Gly 945                 9 #50                 9 #55                 9 #60 aac gaa act gga ctc att act cca aat aaa ca #t gga ctt tta caa aac     3047 Asn Glu Thr Gly Leu Ile Thr Pro Asn Lys Hi #s Gly Leu Leu Gln Asn                 965   #               970   #               975 cca tat cgt ata cca cca ctt ttt ccc atc aa #g tca ttt gtt aaa act     3095 Pro Tyr Arg Ile Pro Pro Leu Phe Pro Ile Ly #s Ser Phe Val Lys Thr             980       #           985       #           990 aaa tgt aag aaa aat ctg cta gag gaa aac tt #t gag gaa cat tca atg     3143 Lys Cys Lys Lys Asn Leu Leu Glu Glu Asn Ph #e Glu Glu His Ser Met         995           #       1000           #      1005 tca cct gaa aga gaa atg gga aat gag aac at #t cca agt aca gtg agc     3191 Ser Pro Glu Arg Glu Met Gly Asn Glu Asn Il #e Pro Ser Thr Val Ser     1010              #   1015               #  1020 aca att agc cgt aat aac att aga gaa aat gt #t ttt aaa gaa gcc agc     3239 Thr Ile Ser Arg Asn Asn Ile Arg Glu Asn Va #l Phe Lys Glu Ala Ser 1025                1030 #                1035  #               1040 tca agc aat att aat gaa gta ggt tcc agt ac #t aat gaa gtg ggc tcc     3287 Ser Ser Asn Ile Asn Glu Val Gly Ser Ser Th #r Asn Glu Val Gly Ser                 1045  #               1050   #              1055 agt att aat gaa ata ggt tcc agt gat gaa aa #c att caa gca gaa cta     3335 Ser Ile Asn Glu Ile Gly Ser Ser Asp Glu As #n Ile Gln Ala Glu Leu             1060      #           1065       #          1070 ggt aga aac aga ggg cca aaa ttg aat gct at #g ctt aga tta ggg gtt     3383 Gly Arg Asn Arg Gly Pro Lys Leu Asn Ala Me #t Leu Arg Leu Gly Val         1075          #       1080           #      1085 ttg caa cct gag gtc tat aaa caa agt ctt cc #t gga agt aat tgt aag     3431 Leu Gln Pro Glu Val Tyr Lys Gln Ser Leu Pr #o Gly Ser Asn Cys Lys     1090              #   1095               #  1100 cat cct gaa ata aaa aag caa gaa tat gaa ga #a gta gtt cag act gtt     3479 His Pro Glu Ile Lys Lys Gln Glu Tyr Glu Gl #u Val Val Gln Thr Val 1105                1110 #                1115  #               1120 aat aca gat ttc tct cca tat ctg att tca ga #t aac tta gaa cag cct     3527 Asn Thr Asp Phe Ser Pro Tyr Leu Ile Ser As #p Asn Leu Glu Gln Pro                 1125  #               1130   #              1135 atg gga agt agt cat gca tct cag gtt tgt tc #t gag aca cct gat gac     3575 Met Gly Ser Ser His Ala Ser Gln Val Cys Se #r Glu Thr Pro Asp Asp             1140      #           1145       #          1150 ctg tta gat gat ggt gaa ata aag gaa gat ac #t agt ttt gct gaa aat     3623 Leu Leu Asp Asp Gly Glu Ile Lys Glu Asp Th #r Ser Phe Ala Glu Asn         1155          #       1160           #      1165 gac att aag gaa agt tct gct gtt ttt agc aa #a agc gtc cag aaa gga     3671 Asp Ile Lys Glu Ser Ser Ala Val Phe Ser Ly #s Ser Val Gln Lys Gly     1170              #   1175               #  1180 gag ctt agc agg agt cct agc cct ttc acc ca #t aca cat ttg gct cag     3719 Glu Leu Ser Arg Ser Pro Ser Pro Phe Thr Hi #s Thr His Leu Ala Gln 1185                1190 #                1195  #               1200 ggt tac cga aga ggg gcc aag aaa tta gag tc #c tca gaa gag aac tta     3767 Gly Tyr Arg Arg Gly Ala Lys Lys Leu Glu Se #r Ser Glu Glu Asn Leu                 1205  #               1210   #              1215 tct agt gag gat gaa gag ctt ccc tgc ttc ca #a cac ttg tta ttt ggt     3815 Ser Ser Glu Asp Glu Glu Leu Pro Cys Phe Gl #n His Leu Leu Phe Gly             1220      #           1225       #          1230 aaa gta aac aat ata cct tct cag tct act ag #g cat agc acc gtt gct     3863 Lys Val Asn Asn Ile Pro Ser Gln Ser Thr Ar #g His Ser Thr Val Ala         1235          #       1240           #      1245 acc gag tgt ctg tct aag aac aca gag gag aa #t tta tta tca ttg aag     3911 Thr Glu Cys Leu Ser Lys Asn Thr Glu Glu As #n Leu Leu Ser Leu Lys     1250              #   1255               #  1260 aat agc tta aat gac tgc agt aac cag gta at #a ttg gca aag gca tct     3959 Asn Ser Leu Asn Asp Cys Ser Asn Gln Val Il #e Leu Ala Lys Ala Ser 1265                1270 #                1275  #               1280 cag gaa cat cac ctt agt gag gaa aca aaa tg #t tct gct agc ttg ttt     4007 Gln Glu His His Leu Ser Glu Glu Thr Lys Cy #s Ser Ala Ser Leu Phe                 1285  #               1290   #              1295 tct tca cag tgc agt gaa ttg gaa gac ttg ac #t gca aat aca aac acc     4055 Ser Ser Gln Cys Ser Glu Leu Glu Asp Leu Th #r Ala Asn Thr Asn Thr             1300      #            1305      #           1310 cag gat cct ttc ttg att ggt tct tcc aaa ca #a atg agg cat cag tct     4103 Gln Asp Pro Phe Leu Ile Gly Ser Ser Lys Gl #n Met Arg His Gln Ser         1315          #       1320           #      1325 gaa agc cag gga gtt ggt ctg agt gac aag ga #a ttg gtt tca gat gat     4151 Glu Ser Gln Gly Val Gly Leu Ser Asp Lys Gl #u Leu Val Ser Asp Asp     1330              #   1335               #  1340 gaa gaa aga gga acg ggc ttg gaa gaa aat aa #t caa gaa gag caa agc     4199 Glu Glu Arg Gly Thr Gly Leu Glu Glu Asn As #n Gln Glu Glu Gln Ser 1345                1350 #                1355  #               1360 atg gat tca aac tta ggt gaa gca gca tct gg #g tgt gag agt gaa aca     4247 Met Asp Ser Asn Leu Gly Glu Ala Ala Ser Gl #y Cys Glu Ser Glu Thr                 1365  #               1370   #              1375 agc gtc tct gaa gac tgc tca ggg cta tcc tc #t cag agt gac att tta     4295 Ser Val Ser Glu Asp Cys Ser Gly Leu Ser Se #r Gln Ser Asp Ile Leu             1380      #           1385       #          1390 acc act cag cag agg gat acc atg caa cat aa #c ctg ata aag ctc cag     4343 Thr Thr Gln Gln Arg Asp Thr Met Gln His As #n Leu Ile Lys Leu Gln         1395          #       1400           #      1405 cag gaa atg gct gaa cta gaa gct gtg tta ga #a cag cat ggg agc cag     4391 Gln Glu Met Ala Glu Leu Glu Ala Val Leu Gl #u Gln His Gly Ser Gln     1410              #   1415               #  1420 cct tct aac agc tac cct tcc atc ata agt ga #c tct tct gcc ctt gag     4439 Pro Ser Asn Ser Tyr Pro Ser Ile Ile Ser As #p Ser Ser Ala Leu Glu 1425                1430 #                1435  #               1440 gac ctg cga aat cca gaa caa agc aca tca ga #a aaa gca gta tta act     4487 Asp Leu Arg Asn Pro Glu Gln Ser Thr Ser Gl #u Lys Val Leu Gln Thr                 1445  #               1450   #              1455 tca cag aaa agt agt gaa tac cct ata agc ca #g aat cca gaa ggc ctt     4535 Ser Gln Lys Ser Ser Glu Tyr Pro Ile Ser Gl #n Asn Pro Glu Gly Xaa             1460      #           1465       #          1470 tct gct gac aag ttt gag gtg tct gca gat ag #t tct acc agt aaa aat     4583 Ser Ala Asp Lys Phe Glu Val Ser Ala Asp Se #r Ser Thr Ser Lys Asn         1475          #       1480           #      1485 aaa gaa cca gga gtg gaa agg tca tcc cct tc #t aaa tgc cca tca tta     4631 Lys Glu Pro Gly Val Glu Arg Ser Ser Pro Se #r Lys Cys Pro Ser Leu     1490              #   1495               #  1500 gat gat agg tgg tac atg cac agt tgc tct gg #g agt ctt cag aat aga     4679 Asp Asp Arg Trp Tyr Met His Ser Cys Ser Gl #y Ser Leu Gln Asn Arg 1505                1510 #                1515  #               1520 aac tac cca tct caa gag gag ctc att aag gt #t gtt gat gtg gag gag     4727 Asn Tyr Pro Pro Gln Glu Glu Leu Ile Lys Va #l Val Asp Val Glu Glu                 1525  #               1530   #              1535 caa cag ctg gaa gag tct ggg cca cac gat tt #g acg gaa aca tct tac     4775 Gln Gln Leu Glu Glu Ser Gly Pro His Asp Le #u Thr Glu Thr Ser Tyr             1540      #           1545       #          1550 ttg cca agg caa gat cta gag gga acc cct ta #c ctg gaa tct gga atc     4823 Leu Pro Arg Gln Asp Leu Glu Gly Thr Pro Ty #r Leu Glu Ser Gly Ile         1555          #       1560           #      1565 agc ctc ttc tct gat gac cct gaa tct gat cc #t tct gaa gac aga gcc     4871 Ser Leu Phe Ser Asp Asp Pro Glu Ser Asp Pr #o Ser Glu Asp Arg Ala     1570              #   1575               #  1580 cca gag tca gct cgt gtt ggc aac ata cca tc #t tca acc tct gca ttg     4919 Pro Glu Ser Ala Arg Val Gly Asn Ile Pro Se #r Ser Thr Ser Ala Leu 1585                1590 #                1595  #               1600 aaa gtt ccc caa ttg aaa gtt gca gaa tct gc #c cag agt cca gct gct     4967 Lys Val Pro Gln Leu Lys Val Ala Glu Ser Al #a Gln Ser Pro Ala Ala                 1605  #               1610   #              1615 gct cat act act gat act gct ggg tat aat gc #a atg gaa gaa agt gtg     5015 Ala His Thr Thr Asp Thr Ala Gly Tyr Asn Al #a Met Glu Glu Ser Val             1620      #           1625       #          1630 agc agg gag aag cca gaa ttg aca gct tca ac #a gaa agg gtc aac aaa     5063 Ser Arg Glu Lys Pro Glu Leu Thr Ala Ser Th #r Glu Arg Val Asn Lys         1635          #       1640           #      1645 aga atg tcc atg gtg gtg tct ggc ctg acc cc #a gaa gaa ttt atg ctc     5111 Arg Met Ser Met Val Val Ser Gly Leu Thr Pr #o Glu Glu Phe Met Leu     1650              #   1655               #  1660 gtg tac aag ttt gcc aga aaa cac cac atc ac #t tta act aat cta att     5159 Val Tyr Lys Phe Ala Arg Lys His His Ile Th #r Leu Thr Asn Leu Ile 1665                1670 #                1675  #               1680 act gaa gag act act cat gtt gtt atg aaa ac #a gat gct gag ttt gtg     5207 Thr Glu Glu Thr Thr His Val Val Met Lys Th #r Asp Ala Glu Phe Val                 1685  #               1690   #              1695 tgt gaa cgg aca ctg aaa tat ttt cta gga at #t gcg gga gga aaa tgg     5255 Cys Glu Arg Thr Leu Lys Tyr Phe Leu Gly Il #e Ala Gly Gly Lys Trp             1700      #           1705       #          1710 gta gtt agc tat ttc tgg gtg acc cag tct at #t aaa gaa aga aaa atg     5303 Val Val Ser Tyr Phe Trp Val Thr Gln Ser Il #e Lys Glu Arg Lys Met         1715          #       1720           #      1725 ctg aat gag cat gat ttt gaa gtc aga gga ga #t gtg gtc aat gga aga     5351 Leu Asn Glu His Asp Phe Glu Val Arg Gly As #p Val Val Asn Gly Arg     1730              #   1735               #  1740 aac cac caa ggt cca aag cga gca aga gaa tc #c cag gac aga aag atc     5399 Asn His Gln Gly Pro Lys Arg Ala Arg Glu Se #r Gln Asp Arg Lys Ile 1745                1750 #                1755  #               1760 ttc agg ggg cta gaa atc tgt tgc tat ggg cc #c ttc acc aac atg ccc     5447 Phe Arg Gly Leu Glu Ile Cys Cys Tyr Gly Pr #o Phe Thr Asn Met Pro                 1765  #               1770   #              1775 aca gat caa ctg gaa tgg atg gta cag ctg tg #t ggt gct tct gtg gtg     5495 Thr Asp Gln Leu Glu Trp Met Val Gln Leu Cy #s Gly Ala Ser Val Val             1780      #          1785        #          1790 aag gag ctt tca tca ttc acc ctt ggc aca gg #t gtc cac cca att gtg     5543 Lys Glu Leu Ser Ser Phe Thr Leu Gly Thr Gl #y Val His Pro Ile Val         1795          #       1800           #      1805 gtt gtg cag cca gat gcc tgg aca gag gac aa #t ggc ttc cat gca att     5591 Val Val Gln Pro Asp Ala Trp Thr Glu Asp As #n Gly Phe His Ala Ile     1810              #   1815               #  1820 ggg cag atg tgt gag gca cct gtg gtg acc cg #a gag tgg gtg ttg gac     5639 Gly Gln Met Cys Glu Ala Pro Val Val Thr Ar #g Glu Trp Val Leu Asp 1825                1830 #                1835  #               1840 agt gta gca ctc tac cag tgc cag gag ctg ga #c acc tac ctg ata ccc     5687 Ser Val Ala Leu Tyr Gln Cys Gln Glu Leu As #p Thr Tyr Leu Ile Pro                 1845  #               1850   #              1855 cag atc ccc cac agc cac tac tgat      #                   #             5712 Gln Ile Pro His Ser His Tyr             1860 (2) INFORMATION FOR SEQ ID NO:2:      (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 1863           (B) TYPE: amino acid           (C) STRANDEDNESS: single           (D) TOPOLOGY: unknown      (x) PUBLICATION INFORMATION:           (A) AUTHORS:  Miki,  #Y., et. al.           (B) TITLE: A strong  #candidate gene for the breast and                ovarian c #ancer susceptibility gene                BRCA1.           (C) JOURNAL: Science           (D) VOLUME: 266           (E) PAGES: 66-71           (F) DATE: 1994           (K) RELEVANT RESIDUES I #N SEQ ID NO:2:  granin box domain                at amino  #acids 1214-1223     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met Asp Leu Ser Ala Leu Arg Val Glu Glu Va #l Gln Asn Val Ile Asn 1               5    #                10   #                15 Ala Met Gln Lys Ile Leu Glu Cys Pro Ile Cy #s Leu Glu Leu Ile Lys             20       #            25       #            30 Glu Pro Val Ser Thr Lys Cys Asp His Ile Ph #e Cys Lys Phe Cys Met         35           #        40           #        45 Leu Lys Leu Leu Asn Gln Lys Lys Gly Pro Se #r Gln Cys Pro Leu Cys     50               #    55               #    60 Lys Asn Asp Ile Thr Lys Arg Ser Leu Gln Gl #u Ser Thr Arg Phe Ser 65                   #70                   #75                   #80 Gln Leu Val Glu Glu Leu Leu Lys Ile Ile Cy #s Ala Phe Gln Leu Asp                 85   #                90   #                95 Thr Gly Leu Glu Tyr Ala Asn Ser Tyr Asn Ph #e Ala Lys Lys Glu Asn             100       #           105       #           110 Asn Ser Pro Glu His Leu Lys Asp Glu Val Se #r Ile Ile Gln Ser Met         115           #       120           #       125 Gly Tyr Arg Asn Arg Ala Lys Arg Leu Leu Gl #n Ser Glu Pro Glu Asn     130               #   135               #   140 Pro Ser Leu Gln Glu Thr Ser Leu Ser Val Gl #n Leu Ser Asn Leu Gly 145                 1 #50                 1 #55                 1 #60 Thr Val Arg Thr Leu Arg Thr Lys Gln Arg Il #e Gln Pro Gln Lys Thr                 165   #               170   #               175 Ser Val Tyr Ile Glu Leu Gly Ser Asp Ser Se #r Glu Asp Thr Val Asn             180       #           185       #           190 Lys Ala Thr Tyr Cys Ser Val Gly Asp Gln Gl #u Leu Leu Gln Ile Thr         195           #       200           #       205 Pro Gln Gly Thr Arg Asp Glu Ile Ser Leu As #p Ser Ala Lys Lys Ala     210               #   215               #   220 Ala Cys Glu Phe Ser Glu Thr Asp Val Thr As #n Thr Glu His His Gln 225                 2 #30                 2 #35                 2 #40 Pro Ser Asn Asn Asp Leu Asn Thr Thr Glu Ly #s Arg Ala Ala Glu Arg                 245   #               250   #               255 His Pro Glu Lys Tyr Gln Gly Ser Ser Val Se #r Asn Leu His Val Glu             260       #           265       #           270 Pro Cys Gly Thr Asn Thr His Ala Ser Ser Le #u Gln His Glu Asn Ser         275           #       280           #       285 Ser Leu Leu Leu Thr Lys Asp Arg Met Asn Va #l Glu Lys Ala Glu Phe     290               #   295               #   300 Cys Asn Lys Ser Lys Gln Pro Gly Leu Ala Ar #g Ser Gln His Asn Arg 305                 3 #10                 3 #15                 3 #20 Trp Ala Gly Ser Lys Glu Thr Cys Asn Asp Ar #g Arg Thr Pro Ser Thr                 325   #               330   #               335 Glu Lys Lys Val Asp Leu Asn Ala Asp Pro Le #u Cys Glu Arg Lys Glu             340       #           345       #           350 Trp Asn Lys Gln Lys Leu Pro Cys Ser Glu As #n Pro Arg Asp Thr Glu         355           #       360           #       365 Asp Val Pro Trp Ile Thr Leu Asn Ser Ser Il #e Gln Lys Val Asn Glu     370               #   375               #   380 Trp Phe Ser Arg Ser Asp Glu Leu Leu Gly Se #r Asp Asp Ser His Asp 385                 3 #90                 3 #95                 4 #00 Gly Glu Ser Glu Ser Asn Ala Lys Val Ala As #p Val Leu Asp Val Leu                 405   #               410   #               415 Asn Glu Val Asp Glu Tyr Ser Gly Ser Ser Gl #u Lys Ile Asp Leu Leu             420       #           425       #           430 Ala Ser Asp Pro His Glu Ala Leu Ile Cys Ly #s Ser Asp Arg Val His         435           #       440           #       445 Ser Lys Ser Val Glu Ser Asp Ile Glu Asp Ly #s Ile Phe Gly Lys Thr     450               #   455               #   460 Tyr Arg Lys Lys Ala Ser Leu Pro Asn Leu Se #r His Val Thr Glu Asn 465                 4 #70                 4 #75                 4 #80 Leu Ile Ile Gly Ala Phe Val Ser Glu Pro Gl #n Ile Ile Gln Glu Arg                 485   #               490   #               495 Pro Leu Thr Asn Lys Leu Lys Arg Lys Arg Ar #g Pro Thr Ser Gly Leu             500       #           505       #           510 His Pro Glu Asp Phe Ile Lys Lys Ala Asp Le #u Ala Val Gln Lys Thr         515           #       520           #       525 Pro Glu Met Ile Asn Gln Gly Thr Asn Gln Th #r Glu Gln Asn Gly Gln     530               #   535               #   540 Val Met Asn Ile Thr Asn Ser Gly His Glu As #n Lys Thr Lys Gly Asp 545                 5 #50                 5 #55                 5 #60 Ser Ile Gln Asn Glu Lys Asn Pro Asn Pro Il #e Glu Ser Leu Glu Lys                 565   #               570   #               575 Glu Ser Ala Phe Lys Thr Lys Ala Glu Pro Il #e Ser Ser Ser Ile Ser             580       #           585       #           590 Asn Glu Leu Glu Leu Asn Ile Met His Asn Se #r Lys Ala Pro Lys Lys         595           #       600           #       605 Asn Arg Leu Arg Arg Lys Ser Ser Thr Arg Hi #s Ile His Ala Leu Glu     610               #   615               #   620 Leu Val Val Ser Arg Asn Leu Ser Pro Pro As #n Cys Thr Glu Leu Gln 625                 6 #30                 6 #35                 6 #40 Ile Asp Ser Cys Ser Ser Ser Glu Glu Ile Ly #s Lys Lys Lys Tyr Asn                 645   #               650   #               655 Gln Met Pro Val Arg His Ser Arg Asn Leu Gl #n Leu Met Glu Gly Lys             660       #           665       #           670 Glu Pro Ala Thr Gly Ala Lys Lys Ser Asn Ly #s Pro Asn Glu Gln Thr         675           #       680           #       685 Ser Lys Arg His Asp Ser Asp Thr Phe Pro Gl #u Leu Lys Leu Thr Asn     690               #   695               #   700 Ala Pro Gly Ser Phe Thr Lys Cys Ser Asn Th #r Ser Glu Leu Lys Glu 705                 7 #10                 7 #15                 7 #20 Phe Val Asn Pro Ser Leu Pro Arg Glu Glu Ly #s Glu Glu Lys Leu Glu                 725   #               730   #               735 Thr Val Lys Val Ser Asn Asn Ala Glu Asp Pr #o Lys Asp Leu Met Leu             740       #           745       #           750 Ser Gly Glu Arg Val Leu Gln Thr Glu Arg Se #r Val Glu Ser Ser Ser         755           #       760           #       765 Ile Ser Leu Val Pro Gly Thr Asp Tyr Gly Th #r Gln Glu Ser Ile Ser     770               #   775               #   780 Leu Leu Glu Val Ser Thr Leu Gly Lys Ala Ly #s Thr Glu Pro Asn Lys 785                 7 #90                 7 #95                 8 #00 Cys Val Ser Gln Cys Ala Ala Phe Glu Asn Pr #o Lys Gly Leu Ile His                 805   #               810   #               815 Gly Cys Ser Lys Asp Asn Arg Asn Asp Thr Gl #u Gly Phe Lys Tyr Pro             820       #           825       #           830 Leu Gly His Glu Val Asn His Ser Arg Glu Th #r Ser Ile Glu Met Glu         835           #       840           #       845 Glu Ser Glu Leu Asp Ala Gln Tyr Leu Gln As #n Thr Phe Lys Val Ser     850               #   855               #   860 Lys Arg Gln Ser Phe Ala Pro Phe Ser Asn Pr #o Gly Asn Ala Glu Glu 865                 8 #70                 8 #75                 8 #80 Glu Cys Ala Thr Phe Ser Ala His Ser Gly Se #r Leu Lys Lys Gln Ser                 885   #               890   #               895 Pro Lys Val Thr Phe Glu Cys Glu Gln Lys Gl #u Glu Asn Gln Gly Lys             900       #           905       #          910 Asn Glu Ser Asn Ile Lys Pro Val Gln Thr Va #l Asn Ile Thr Ala Gly         915           #       920           #       925 Phe Pro Val Val Gly Gln Lys Asp Lys Pro Va #l Asp Asn Ala Lys Cys     930               #   935               #   940 Ser Ile Lys Gly Gly Ser Arg Phe Cys Leu Se #r Ser Gln Phe Arg Gly 945                 9 #50                 9 #55                 9 #60 Asn Glu Thr Gly Leu Ile Thr Pro Asn Lys Hi #s Gly Leu Leu Gln Asn                 965   #              970    #              975 Pro Tyr Arg Ile Pro Pro Leu Phe Pro Ile Ly #s Ser Phe Val Lys Thr             980       #           985       #           990 Lys Cys Lys Lys Asn Leu Leu Glu Glu Asn Ph #e Glu Glu His Ser Met         995           #       1000           #      1005 Ser Pro Glu Arg Glu Met Gly Asn Glu Asn Il #e Pro Ser Thr Val Ser     1010              #   1015               #  1020 Thr Ile Ser Arg Asn Asn Ile Arg Glu Asn Va #l Phe Lys Glu Ala Ser 1025                1030 #                1035  #               1040 Ser Ser Asn Ile Asn Glu Val Gly Ser Ser Th #r Asn Glu Val Gly Ser                 1045  #               1050   #              1055 Ser Ile Asn Glu Ile Gly Ser Ser Asp Glu As #n Ile Gln Ala Glu Leu             1060      #           1065       #          1070 Gly Arg Asn Arg Gly Pro Lys Leu Asn Ala Me #t Leu Arg Leu Gly Val         1075          #       1080           #      1085 Leu Gln Pro Glu Val Tyr Lys Gln Ser Leu Pr #o Gly Ser Asn Cys Lys     1090              #   1095               #  1100 His Pro Glu Ile Lys Lys Gln Glu Tyr Glu Gl #u Val Val Gln Thr Val 1105                1110 #                1115  #               1120 Asn Thr Asp Phe Ser Pro Tyr Leu Ile Ser As #p Asn Leu Glu Gln Pro                 1125  #              1130    #             1135 Met Gly Ser Ser His Ala Ser Gln Val Cys Se #r Glu Thr Pro Asp Asp             1140      #           1145       #          1150 Leu Leu Asp Asp Gly Glu Ile Lys Glu Asp Th #r Ser Phe Ala Glu Asn         1155          #       1160           #      1165 Asp Ile Lys Glu Ser Ser Ala Val Phe Ser Ly #s Ser Val Gln Lys Gly     1170              #   1175               #  1180 Glu Leu Ser Arg Ser Pro Ser Pro Phe Thr Hi #s Thr His Leu Ala Gln 1185                1190 #                1195  #               1200 Gly Tyr Arg Arg Gly Ala Lys Lys Leu Glu Se #r Ser Glu Glu Asn Leu                 1205  #               1210   #              1215 Ser Ser Glu Asp Glu Glu Leu Pro Cys Phe Gl #n His Leu Leu Phe Gly             1220      #           1225       #          1230 Lys Val Asn Asn Ile Pro Ser Gln Ser Thr Ar #g His Ser Thr Val Ala         1235          #       1240           #      1245 Thr Glu Cys Leu Ser Lys Asn Thr Glu Glu As #n Leu Leu Ser Leu Lys     1250              #   1255               #  1260 Asn Ser Leu Asn Asp Cys Ser Asn Gln Val Il #e Leu Ala Lys Ala Ser 1265                1270 #                1275  #               1280 Gln Glu His His Leu Ser Glu Glu Thr Lys Cy #s Ser Ala Ser Leu Phe                 1285  #               1290   #              1295 Ser Ser Gln Cys Ser Glu Leu Glu Asp Leu Th #r Ala Asn Thr Asn Thr             1300      #           1305       #          1310 Gln Asp Pro Phe Leu Ile Gly Ser Ser Lys Gl #n Met Arg His Gln Ser         1315          #       1320           #      1325 Glu Ser Gln Gly Val Gly Leu Ser Asp Lys Gl #u Leu Val Ser Asp Asp     1330              #   1335               #  1340 Glu Glu Arg Gly Thr Gly Leu Glu Glu Asn As #n Gln Glu Glu Gln Ser 1345                1350 #                1355  #               1360 Met Asp Ser Asn Leu Gly Glu Ala Ala Ser Gl #y Cys Glu Ser Glu Thr                 1365  #               1370   #              1375 Ser Val Ser Glu Asp Cys Ser Gly Leu Ser Se #r Gln Ser Asp Ile Leu             1380      #           1385       #          1390 Thr Thr Gln Gln Arg Asp Thr Met Gln His As #n Leu Ile Lys Leu Gln         1395          #       1400           #      1405 Gln Glu Met Ala Glu Leu Glu Ala Val Leu Gl #u Gln His Gly Ser Gln     1410              #   1415               #  1420 Pro Ser Asn Ser Tyr Pro Ser Ile Ile Ser As #p Ser Ser Ala Leu Glu 1425                1430 #                1435  #               1440 Asp Leu Arg Asn Pro Glu Gln Ser Thr Ser Gl #u Lys Val Leu Gln Thr                 1445  #               1450   #              1455 Ser Gln Lys Ser Ser Glu Tyr Pro Ile Ser Gl #n Asn Pro Glu Gly Xaa             1460      #           1465       #          1470 Ser Ala Asp Lys Phe Glu Val Ser Ala Asp Se #r Ser Thr Ser Lys Asn         1475          #       1480           #      1485 Lys Glu Pro Gly Val Glu Arg Ser Ser Pro Se #r Lys Cys Pro Ser Leu     1490              #   1495               #  1500 Asp Asp Arg Trp Tyr Met His Ser Cys Ser Gl #y Ser Leu Gln Asn Arg 1505                1510 #                1515  #               1520 Asn Tyr Pro Pro Gln Glu Glu Leu Ile Lys Va #l Val Asp Val Glu Glu                 1525  #               1530   #              1535 Gln Gln Leu Glu Glu Ser Gly Pro His Asp Le #u Thr Glu Thr Ser Tyr             1540      #           1545       #          1550 Leu Pro Arg Gln Asp Leu Glu Gly Thr Pro Ty #r Leu Glu Ser Gly Ile         1555          #       1560           #      1565 Ser Leu Phe Ser Asp Asp Pro Glu Ser Asp Pr #o Ser Glu Asp Arg Ala     1570              #   1575               #  1580 Pro Glu Ser Ala Arg Val Gly Asn Ile Pro Se #r Ser Thr Ser Ala Leu 1585                1590 #                1595  #               1600 Lys Val Pro Gln Leu Lys Val Ala Glu Ser Al #a Gln Ser Pro Ala Ala                 1605  #               1610   #              1615 Ala His Thr Thr Asp Thr Ala Gly Tyr Asn Al #a Met Glu Glu Ser Val             1620      #           1625       #          1630 Ser Arg Glu Lys Pro Glu Leu Thr Ala Ser Th #r Glu Arg Val Asn Lys         1635          #       1640           #      1645 Arg Met Ser Met Val Val Ser Gly Leu Thr Pr #o Glu Glu Phe Met Leu     1650              #   1655               #  1660 Val Tyr Lys Phe Ala Arg Lys His His Ile Th #r Leu Thr Asn Leu Ile 1665                1670 #                1675  #               1680 Thr Glu Glu Thr Thr His Val Val Met Lys Th #r Asp Ala Glu Phe Val                 1685  #               1690   #              1695 Cys Glu Arg Thr Leu Lys Tyr Phe Leu Gly Il #e Ala Gly Gly Lys Trp             1700      #           1705       #          1710 Val Val Ser Tyr Phe Trp Val Thr Gln Ser Il #e Lys Glu Arg Lys Met         1715          #       1720           #      1725 Leu Asn Glu His Asp Phe Glu Val Arg Gly As #p Val Val Asn Gly Arg     1730              #   1735               #  1740 Asn His Gln Gly Pro Lys Arg Ala Arg Glu Se #r Gln Asp Arg Lys Ile 1745                1750 #                1755  #               1760 Phe Arg Gly Leu Glu Ile Cys Cys Tyr Gly Pr #o Phe Thr Asn Met Pro                 1765  #               1770   #              1775 Thr Asp Gln Leu Glu Trp Met Val Gln Leu Cy #s Gly Ala Ser Val Val             1780      #          1785        #          1790 Lys Glu Leu Ser Ser Phe Thr Leu Gly Thr Gl #y Val His Pro Ile Val         1795          #       1800           #      1805 Val Val Gln Pro Asp Ala Trp Thr Glu Asp As #n Gly Phe His Ala Ile     1810              #   1815               #  1820 Gly Gln Met Cys Glu Ala Pro Val Val Thr Ar #g Glu Trp Val Leu Asp 1825                1830 #                1835  #               1840 Ser Val Ala Leu Tyr Gln Cys Gln Glu Leu As #p Thr Tyr Leu Ile Pro                 1845  #               1850   #              1855 Gln Ile Pro His Ser His Tyr             1860 (2) INFORMATION FOR SEQ ID NO:3:      (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 11283           (B) TYPE: nucleic acid           (C) STRANDEDNESS: double           (D) TOPOLOGY: linear     (ix) FEATURE:           (A) NAME/KEY: BRCA2      (x) PUBLICATION INFORMATION:           (A) AUTHORS:      #Wooster, R. et al.           (B) TITLE:  Identificat #ion of the breast cancer                susceptabili #ty gene BRCA2           (C) JOURNAL:      #Nature           (D) VOLUME:      # 379           (E) PAGES:      #  789-792           (F) DATE:      #   1995     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: #3: ggcggagccg ctgtggcact gctgcgcctc tgctgcgcct cgggtgtctt tt #gcggcggt     60 gggtcgccgc cgggagaagc gtgaggggac agatttgtga ccggcgcggt tt #ttgtcagc    120 ttactccggc caaaaaagaa ctgcacctct ggagcggact tatttaccaa gc #attggagg    180 aatatcgtag gtaaaa              #                   #                   #   196 atg cct att gga tcc aaa gag agg cca aca tt #t ttt gaa att ttt aag      244 Met Pro Ile Gly Ser Lys Glu Arg Pro Thr Ph #e Phe Glu Ile Phe Lys 1               5    #                10   #                15 aca cgc tgc aac aaa gca gat tta gga cca at #a agt ctt aat tgg ttt      292 Thr Arg Cys Asn Lys Ala Asp Leu Gly Pro Il #e Ser Leu Asn Trp Phe 20                   #25                   #30 gaa gaa ctt tct tca gaa gct cca ccc tat aa #t tct gaa cct gca gaa      340 Glu Glu Leu Ser Ser Glu Ala Pro Pro Tyr As #n Ser Glu Pro Ala Glu 35                   #40                   #45 gaa tct gaa cat aaa aac aac aat tac gaa cc #a aac cta ttt aaa act      388 Glu Ser Glu His Lys Asn Asn Asn Tyr Glu Pr #o Asn Leu Phe Lys Thr 50                   #55                   #60 cca caa agg aaa cca tct tat aat cag ctg gc #t tca act cca ata ata      436 Pro Gln Arg Lys Pro Ser Tyr Asn Gln Leu Al #a Ser Thr Pro Ile Ile 65                   #70                   #75                   #80 ttc aaa gag caa ggg ctg act ctg ccg ctg ta #c caa tct cct gta aaa      484 Phe Lys Glu Gln Gly Leu Thr Leu Pro Leu Ty #r Gln Ser Pro Val Lys 85                   #90                   #95 gaa tta gat aaa ttc aaa tta gac tta gga ag #g aat gtt ccc aat agt      532 Glu Leu Asp Lys Phe Lys Leu Asp Leu Gly Ar #g Asn Val Pro Asn Ser 100                 1 #05                 1 #10 aga cat aaa agt ctt cgc aca gtg aaa act aa #a atg gat caa gca gat      580 Arg His Lys Ser Leu Arg Thr Val Lys Tyr Ly #s Met Asp Gln Ala Asp 115                 1 #20                 1 #25 gat gtt tcc tgt cca ctt cta aat tct tgt ct #t agt gaa agt cct gtt      628 Asp Val Ser Cys Pro Leu Leu Asn Ser Cys Le #u Ser Glu Ser Pro Val 130                 1 #35                 1 #40 gtt cta caa tgt aca cat gta aca cca caa ag #a gat aag tca gtg gta      676 Val Leu Gln Cys Thr His Val Thr Pro Gln Ar #g Asp Lys Ser Val Val 145                 1 #50                 1 #55                 1 #60 tgt ggg agt ttg ttt cat aca cca aag ttt gt #g aag ggt cgt cag aca      724 Cys Gly Ser Leu Phe His Thr Pro Lys Phe Va #l Lys Gly Arg Gln Thr 165                 1 #70                 1 #75 cca aaa cat att tct gaa agt cta gga gct ga #g gtg gat cct gat atg      772 Pro Lys His Ile Ser Glu Ser Leu Gly Ala Gl #u Val Asp Pro Asp Met 180                 1 #85                 1 #90 tct tgg tca agt tct tta gct aca cca ccc ac #c ctt agt tct act gtg      820 Ser Trp Ser Ser Ser Leu Ala Thr Pro Pro Th #r Leu Ser Ser Thr Val 195                 2 #00                 2 #05 ctc ata gtc aga aat gaa gaa gca tct gaa ac #t gta ttt cct cat gat      868 Leu Ile Val Arg Asn Glu Glu Ala Ser Glu Th #r Val Phe Pro His Asp 210                 2 #15                 2 #20 act act gct aat gtg aaa agc tat ttt tcc aa #t cat gat gaa agt ctg      916 Thr Thr Ala Asn Val Lys Ser Tyr Phe Ser As #n His Asp Glu Ser Leu 225                 2 #30                 2 #35                 2 #40 aag aaa aat gat aga ttt atc gct tct gtg ac #a gac agt gaa aac aca      964 Lys Lys Asn Asp Arg Phe Ile Ala Ser Val Th #r Asp Ser Glu Asn Thr 245                 2 #50                 2 #55 aat caa aga gaa gct gca agt cat gga ttt gg #a aaa aca tca ggg aat     1012 Asn Gln Arg Glu Ala Ala Ser His Gly Phe Gl #y Lys Thr Ser Gly Asn 260                 2 #65                 2 #70 tca ttt aaa gta aat agc tgc aaa gac cac at #t gga aag tca atg cca     1060 Ser Phe Lys Val Asn Ser Cys Lys Asp His Il #e Gly Lys Ser Met Pro 275                 2 #80                 2 #85 aat gtc cta gaa gat gaa gta tat gaa aca gt #t gta gat acc tct gaa     1108 Asn Val Leu Glu Asp Glu Val Tyr Glu Thr Va #l Val Asp Thr Ser Glu 290                 2 #95                 3 #00 gaa gat agt ttt tca tta tgt ttt tct aaa tg #t aga aca aaa aat cta     1156 Glu Asp Ser Phe Ser Leu Cys Phe Ser Lys Cy #s Arg Thr Lys Asn Leu 305                 3 #10                 3 #15                 3 #20 caa aaa gta aga act agc aag act agg aaa aa #a att ttc cat gaa gca     1204 Gln Lys Val Arg Thr Ser Lys Thr Arg Lys Ly #s Ile Phe His Glu Ala 325                 3 #30                 3 #35 aac gct gat gaa tgt gaa aaa tct aaa aac ca #a gtg aaa gaa aaa tac     1252 Asn Ala Asp Glu Cys Glu Lys Ser Lys Asn Gl #n Val Lys Glu Lys Tyr 340                 3 #45                 3 #50 tca ttt gta tct gaa gtg gaa cca aat gat ac #t gat cca tta gat tca     1300 Ser Phe Val Ser Glu Val Glu Pro Asn Asp Th #r Asp Pro Leu Asp Ser 355                 3 #60                 3 #65 aat gta gca cat cag aag ccc ttt gag agt gg #a agt gac aaa atc tcc     1348 Asn Val Ala His Gln Lys Pro Phe Glu Ser Gl #y Ser Asp Lys Ile Ser 370                 3 #75                 3 #80 aag gaa gtt gta ccg tct ttg gcc tgt gaa tg #g tct caa cta acc ctt     1396 Lys Glu Val Val Pro Ser Leu Ala Cys Glu Tr #p Ser Gln Leu Thr Leu 385                 3 #90                 3 #95                 4 #00 tca ggt cta aat gga gcc cag atg gag aaa at #a ccc cta ttg cat att     1444 Ser Gly Leu Asn Gly Ala Gln Met Glu Lys Il #e Pro Leu Leu His Ile 405                 4 #10                 4 #15 tct tca tgt gac caa aat att tca gaa aaa ga #c cta tta gac aca gag     1492 Ser Ser Cys Asp Gln Asn Ile Ser Glu Lys As #p Leu Leu Asp Thr Glu 420                 4 #25                 4 #30 aac aaa aga aag aaa gat ttt ctt act tca ga #g aat tct ttg cca cgt     1540 Asn Lys Arg Lys Lys Asp Phe Leu Thr Ser Gl #u Asn Ser Leu Pro Arg 435                 4 #40                 4 #45 att tct agc cta cca aaa tca gag aag cca tt #a aat gag gaa aca gtg     1588 Ile Ser Ser Leu Pro Lys Ser Glu Lys Pro Le #u Asn Glu Glu Thr Val 450                 4 #55                 4 #60 gta aat aag aga gat gaa gag cag cat ctt ga #a tct cat aca gac tgc     1636 Val Asn Lys Arg Asp Glu Glu Gln His Leu Gl #u Ser His Thr Asp Cys 465                 4 #70                 4 #75                 4 #80 att ctt gca gta aag cag gca ata tct gga ac #t tct cca gtg gct tct     1684 Ile Leu Ala Val Lys Gln Ala Ile Ser Gly Th #r Ser Pro Val Ala Ser 485                 4 #90                 4 #95 tca ttt cag ggt atc aaa aag tct ata ttc ag #a ata aga gaa tca cct     1732 Ser Phe Gln Gly Ile Lys Lys Ser Ile Phe Ar #g Ile Arg Glu Ser Pro 500                 5 #05                 5 #10 aaa gag act ttc aat gca agt ttt tca ggt ca #t atg act gat cca aac     1780 Lys Glu Thr Phe Asn Ala Ser Phe Ser Gly Hi #s Met Thr Asp Pro Asn 515                 5 #20                 5 #25 ttt aaa aaa gaa act gaa gcc tct gaa agt gg #a ctg gaa ata cat act     1828 Phe Lys Lys Glu Thr Glu Ala Ser Glu Ser Gl #y Leu Glu Ile His Thr 530                 5 #35                 5 #40 gtt tgc tca cag aag gag gac tcc tta tgt cc #a aat tta att gat aat     1876 Val Cys Ser Gln Lys Glu Asp Ser Leu Cys Pr #o Asn Leu Ile Asp Asn 545                 5 #50                 5 #55                 5 #60 gga agc tgg cca gcc acc acc aca cag aat tc #t gta gct ttg aag aat     1924 Gly Ser Trp Pro Ala Thr Thr Thr Gln Asn Se #r Val Ala Leu Lys Asn 565                 5 #70                 5 #75 gca ggt tta ata tcc act ttg aaa aag aaa ac #a aat aag ttt att tat     1972 Ala Gly Leu Ile Ser Thr Leu Lys Lys Lys Th #r Asn Lys Phe Ile Tyr 580                 5 #85                 5 #90 gct ata cat gat gaa aca ttt tat aaa gga aa #a aaa ata ccg aaa gac     2020 Ala Ile His Asp Glu Thr Phe Tyr Lys Gly Ly #s Lys Ile Pro Lys Asp 595                 6 #00                 6 #05 caa aaa tca gaa cta att aac tgt tca gcc ca #g ttt gaa gca aat gct     2068 Gln Lys Ser Glu Leu Ile Asn Cys Ser Ala Gl #n Phe Glu Ala Asn Ala 610                 6 #15                 6 #20 ttt gaa gca cca ctt aca ttt gca aat gct ga #t tca ggt tta ttg cat     2116 Phe Glu Ala Pro Leu Thr Phe Ala Asn Ala As #p Ser Gly Leu Leu His 625                 6 #30                 6 #35                 6 #40 tct tct gtg aaa aga agc tgt tca cag aat ga #t tct gaa gaa cca act     2164 Ser Ser Val Lys Arg Ser Cys Ser Gln Asn As #p Ser Glu Glu Pro Thr 645                 6 #50                 6 #55 ttg tcc tta act agc tct ttt ggg aca att ct #g agg aaa tgt tct aga     2212 Leu Ser Leu Thr Ser Ser Phe Gly Thr Ile Le #u Arg Lys Cys Ser Arg 660                 6 #65                 6 #70 aat gaa aca tgt tct aat aat aca gta atc tc #t cag gat ctt gat tat     2260 Asn Glu Thr Cys Ser Asn Asn Thr Val Ile Se #r Gln Asp Leu Asp Tyr 675                 6 #80                 6 #85 aaa gaa gca aaa tgt aat aag gaa aaa cta ca #g tta ttt att acc cca     2308 Lys Glu Ala Lys Cys Asn Lys Glu Lys Leu Gl #n Leu Phe Ile Thr Pro 690                   #  695                #  700 gaa gct gat tct ctg tca tgc ctg cag gaa gg #a cag tgt gaa aat gat     2356 Glu Ala Asp Ser Leu Ser Cys Leu Gln Glu Gl #y Gln Cys Glu Asn Asp 705                 7 #10                 7 #15                 7 #20 cca aaa agc aaa aaa gtt tca gat ata aaa ga #a gag gtc ttg gct gca     2404 Pro Lys Ser Lys Lys Val Ser Asp Ile Lys Gl #u Glu Val Leu Ala Ala 725                 7 #30                 7 #35 gca tgt cac cca gta caa cat tca aaa gtg ga #a tac agt gat act gac     2452 Ala Cys His Pro Val Gln His Ser Lys Val Gl #u Tyr Ser Asp Thr Asp 740                 7 #45                 7 #50 ttt caa tcc cag aaa agt ctt tta tat gat ca #t gaa aat gcc agc act     2500 Phe Gln Ser Gln Lys Ser Leu Leu Tyr Asp Hi #s Glu Asn Ala Ser Thr 755                 7 #60                 7 #65 ctt att tta act cct act tcc aag gat gtt ct #g tca aac cta gtc atg     2548 Leu Ile Leu Thr Pro Thr Ser Lys Asp Val Le #u Ser Asn Leu Val Met 770                 7 #75                 7 #80 att tct aga ggc aaa gaa tca tac aaa atg tc #a gac aag ctc aaa ggt     2596 Ile Ser Arg Gly Lys Glu Ser Tyr Lys Met Se #r Asp Lys Leu Lys Gly 785                 7 #90                 7 #95                 8 #00 aac aat tat gaa tct gat gtt gaa tta acc aa #a aat att ccc atg gaa     2644 Asn Asn Tyr Glu Ser Asp Val Glu Leu Thr Ly #s Asn Ile Pro Met Glu 805                 8 #10                 8 #15 aag aat caa gat gta tgt gct tta aat gaa aa #t tat aaa aac gtt gag     2692 Lys Asn Gln Asp Val Cys Ala Leu Asn Glu As #n Tyr Lys Asn Val Glu 820                 8 #25                 8 #30 ctg ttg cca cct gaa aaa tac atg aga gta gc #a tca cct tca aga aag     2740 Leu Leu Pro Pro Glu Lys Tyr Met Arg Val Al #a Ser Pro Ser Arg Lys 835                 8 #40                 8 #45 gta caa ttc aac caa aac aca aat cta aga gt #a atc caa aaa aat caa     2788 Val Gln Phe Asn Gln Asn Thr Asn Leu Arg Va #l Ile Gln Lys Asn Gln 850                 8 #55                 8 #60 gaa gaa act act tca att tca aaa ata act gt #c aat cca gac tct gaa     2836 Glu Glu Thr Thr Ser Ile Ser Lys Ile Thr Va #l Asn Pro Asp Ser Glu 865                 8 #70                 8 #75                 8 #80 gaa ctt ttc tca gac aat gag aat aat ttt gt #c ttc caa gta gct aat     2884 Glu Leu Phe Ser Asp Asn Glu Asn Asn Phe Va #l Phe Gln Val Ala Asn 885                 8 #90                 8 #95 gaa agg aat aat ctt gct tta gga aat act aa #g gaa ctt cat gaa aca     2932 Glu Arg Asn Asn Leu Ala Leu Gly Asn Thr Ly #s Glu Leu His Glu Thr 900                 9 #05                 9 #10 gac ttg act tgt gta aac gaa ccc att ttc aa #g aac tct acc atg gtt     2980 Asp Leu Thr Cys Val Asn Glu Pro Ile Phe Ly #s Asn Ser Thr Met Val 915                 9 #20                 9 #25 tta tat gga gac aca ggt gat aaa caa gca ac #c caa gtg tca att aaa     3028 Leu Tyr Gly Asp Thr Gly Asp Lys Gln Ala Th #r Gln Val Ser Ile Lys 930                 9 #35                 9 #40 aaa gat ttg gtt tat gtt ctt gca gag gag aa #c aaa aat agt gta aag     3076 Lys Asp Leu Val Tyr Val Leu Ala Glu Glu As #n Lys Asn Ser Val Lys 945                 9 #50                 9 #55                 9 #60 cag cat ata aaa atg act cta ggt caa gat tt #a aaa tcg gac atc tcc     3124 Gln His Ile Lys Met Thr Leu Gly Gln Asp Le #u Lys Ser Asp Ile Ser 965                 9 #70                 9 #75 ttg aat ata gat aaa ata cca gaa aaa aat aa #t gat tac atg aac aaa     3172 Leu Asn Ile Asp Lys Ile Pro Glu Lys Asn As #n Asp Tyr Met Asn Lys 980                 9 #85                 9 #90 tgg gca gga ctc tta ggt cca att tca aat ca #c agt ttt gga ggt agc     3220 Trp Ala Gly Leu Leu Gly Pro Ile Ser Asn Hi #s Ser Phe Gly Gly Ser 995                 1 #000                1005 ttc aga aca gct tca aat aag gaa atc aag ct #c tct gaa cat aac att     3268 Phe Arg Thr Ala Ser Asn Lys Glu Ile Lys Le #u Ser Glu His Asn Ile 1010                1015 #                1020 aag aag agc aaa atg ttc ttc aaa gat att ga #a gaa caa tat cct act     3316 Lys Lys Ser Lys Met Phe Phe Lys Asp Ile Gl #u Glu Gln Tyr Pro Thr 1025                1030 #                1035  #               1040 agt tta gct tgt gtt gaa att gta aat acc tt #g gca tta gat aat caa      3364 Ser Leu Ala Cys Val Glu Ile Val Asn Thr Le #u Ala Leu Asp Asn Gln 1045                1050 #                1055 aag aaa ctg agc aag cct cag tca att aat ac #t gta tct gca cat tta     3412 Lys Lys Leu Ser Lys Pro Gln Ser Ile Asn Th #r Val Ser Ala His Leu 1060                1065 #                1070 cag agt agt gta gtt gtt tct gat tgt aaa aa #t agt cat ata acc cct     3460 Gln Ser Ser Val Val Val Ser Asp Cys Lys As #n Ser His Ile Thr Pro 1075                1080 #                1085 cag atg tta ttt tcc aag cag gat ttt aat tc #a aac cat aat tta aca     3508 Gln Met Leu Phe Ser Lys Gln Asp Phe Asn Se #r Asn His Asn Leu Thr 1090                1095 #                1100 cct agc caa aag gca gaa att aca gaa ctt tc #t act ata tta gaa gaa     3556 Pro Ser Gln Lys Ala Glu Ile Thr Glu Leu Se #r Thr Ile Leu Glu Glu 1105                1110 #                1115  #               1120 tca gga agt cag ttt gaa ttt act cag ttt ag #a aaa cca agc tac ata     3604 Ser Gly Ser Gln Phe Glu Phe Thr Gln Phe Ar #g Lys Pro Ser Tyr Ile 1125                1130 #                1135 ttg cag aag agt aca ttt gaa gtg cct gaa aa #c cag atg act atc tta     3652 Leu Gln Lys Ser Thr Phe Glu Val Pro Glu As #n Gln Met Thr Ile Leu 1140                1145 #                1150 aag acc act tct gag gaa tgc aga gat gct ga #t ctt cat gtc ata atg     3700 Lys Thr Thr Ser Glu Glu Cys Arg Asp Ala As #p Leu His Val Ile Met 1155                1160 #                1165 aat gcc cca tcg att ggt cag gta gac agc ag #c aag caa ttt gaa ggt     3748 Asn Ala Pro Ser Ile Gly Gln Val Asp Ser Se #r Lys Gln Phe Glu Gly 1170                1175 #                1180 aca gtt gaa att aaa cgg aag ttt gct ggc ct #g ttg aaa aat gac tgt     3796 Thr Val Glu Ile Lys Arg Lys Phe Ala Gly Le #u Leu Lys Asn Asp Cys 1185                1190 #                1195  #               1200 aac aaa agt gct tct ggt tat tta aca gat ga #a aat gaa gtg ggg ttt     3844 Asn Lys Ser Ala Ser Gly Tyr Leu Thr Asp Gl #u Asn Glu Val Gly Phe 1205                1210 #                1215 agg ggc ttt tat tct gct cat ggc aca aaa ct #g aat gtt tct act gaa     3892 Arg Gly Phe Tyr Ser Ala His Gly Thr Lys Le #u Asn Val Ser Thr Glu 1220                1225 #                1230 gct ctg caa aaa gct gtg aaa ctg ttt agt ga #t att gag aat att agt     3940 Ala Leu Gln Lys Ala Val Lys Leu Phe Ser As #p Ile Glu Asn Ile Ser 1235                1240 #                1245 gag gaa act tct gca gag gta cat cca ata ag #t tta tct tca agt aaa     3988 Glu Glu Thr Ser Ala Glu Val His Pro Ile Se #r Leu Ser Ser Ser Lys 1250                1255 #                1260 tgt cat gat tct gtt gtt tca atg ttt aag at #a gaa aat cat aat gat     4036 Cys His Asp Ser Val Val Ser Met Phe Lys Il #e Glu Asn His Asn Asp 1265                1270 #                1275  #               1280 aaa act gta agt gaa aaa aat aat aaa tgc ca #a ctg ata tta caa aat     4084 Lys Thr Val Ser Glu Lys Asn Asn Lys Cys Gl #n Leu Ile Leu Gln Asn 1285                1290 #                1295 aat att gaa atg act act ggc act ttt gtt ga #a gaa att act gaa aat     4132 Asn Ile Glu Met Thr Thr Gly Thr Phe Val Gl #u Glu Ile Thr Glu Asn 1300                1305 #                1310 tac aag aga aat act gaa aat gaa gat aac aa #a tat act gct gcc agt     4180 Tyr Lys Arg Asn Thr Glu Asn Glu Asp Asn Ly #s Tyr Thr Ala Ala Ser 1315                1320 #                1325 aga aat tct cat aac tta gaa ttt gat ggc ag #t gat tca agt aaa aat     4228 Arg Asn Ser His Asn Leu Glu Phe Asp Gly Se #r Asp Ser Ser Lys Asn 1330                1335 #                1340 gat act gtt tgt att cat aaa gat gaa acg ga #c ttg cta ttt act gat     4276 Asp Thr Val Cys Ile His Lys Asp Glu Thr As #p Leu Leu Phe Thr Asp 1345                1350 #                1355  #               1360 cag cac aac ata tgt ctt aaa tta tct ggc ca #g ttt atg aag gag gga     4324 Gln His Asn Ile Cys Leu Lys Leu Ser Gly Gl #n Phe Met Lys Glu Gly 1365                1370 #                1375 aac act cag att aaa gaa gat ttg tca gat tt #a act ttt ttg gaa gtt     4372 Asn Thr Gln Ile Lys Glu Asp Leu Ser Asp Le #u Thr Phe Leu Glu Val 1380                1385 #                1390 gcg aaa gct caa gaa gca tgt cat ggt aat ac #t tca aat aaa gaa cag     4420 Ala Lys Ala Gln Glu Ala Cys His Gly Asn Th #r Ser Asn Lys Glu Gln 1395                1400 #                1405 tta act gct act aaa acg gag caa aat ata aa #a gat ttt gag act tct     4468 Leu Thr Ala Thr Lys Thr Glu Gln Asn Ile Ly #s Asp Phe Glu Thr Ser 1410                1415 #                1420 gat aca ttt ttt cag act gca agt ggg aaa aa #t att agt gtc gcc aaa     4516 Asp Thr Phe Phe Gln Thr Ala Ser Gly Lys As #n Ile Ser Val Ala Lys 1425                1430 #                1435  #               1440 gag tta ttt aat aaa att gta aat ttc ttt ga #t cag aaa cca gaa gaa     4564 Glu Leu Phe Asn Lys Ile Val Asn Phe Phe As #p Gln Lys Pro Glu Glu 1445                1450 #                1455 ttg cat aac ttt tcc tta aat tct gaa tta ca #t tct gac ata aga aag     4612 Leu His Asn Phe Ser Leu Asn Ser Glu Leu Hi #s Ser Asp Ile Arg Lys 1460                1465 #                1470 aac aaa atg gac att cta agt tat gag gaa ac #a gac ata gtt aaa cac     4660 Asn Lys Met Asp Ile Leu Ser Tyr Glu Glu Th #r Asp Ile Val Lys His 1475                1480 #                1485 aaa ata ctg aaa gaa agt gtc cca gtt ggt ac #t gga aat caa cta gtg     4708 Lys Ile Leu Lys Glu Ser Val Pro Val Gly Th #r Gly Asn Gln Leu Val 1490                1495 #                1500 acc ttc cag gga caa ccc gaa cgt gat gaa aa #g atc aaa gaa cct act     4756 Thr Phe Gln Gly Gln Pro Glu Arg Asp Glu Ly #s Ile Lys Glu Pro Thr 1505                1510 #                1515  #               1520 ctg ttg ggt ttt cat aca gct agc gga aaa aa #a gtt aaa att gca aag     4804 Leu Leu Gly Phe His Thr Ala Ser Gly Lys Ly #s Val Lys Ile Ala Lys 1525                1530 #                1535 gaa tct ttg gac aaa gtg aaa aac ctt ttt ga #t gaa aaa gag caa ggt     4852 Glu Ser Leu Asp Lys Val Lys Asn Leu Phe As #p Glu Lys Glu Gln Gly 1540                1545 #                1550 act agt gaa atc acc agt ttt agc cat caa tg #g gca aag acc cta aag     4900 Thr Ser Glu Ile Thr Ser Phe Ser His Gln Tr #p Ala Lys Thr Leu Lys 1555                1560 #                1565 tac aga gag gcc tgt aaa gac ctt gaa tta gc #a tgt gag acc att gag     4948 Tyr Arg Glu Ala Cys Lys Asp Leu Glu Leu Al #a Cys Glu Thr Ile Glu 1570                1575 #                1580 atc aca gct gcc cca aag tgt aaa gaa atg ca #g aat tct ctc aat aat     4996 Ile Thr Ala Ala Pro Lys Cys Lys Glu Met Gl #n Asn Ser Leu Asn Asn 1585                1590 #                1595  #               1600 gat aaa aac ctt gtt tct att gag act gtg gt #g cca cct aag ctc tta     5044 Asp Lys Asn Leu Val Ser Ile Glu Thr Val Va #l Pro Pro Lys Leu Leu 1605                1610 #                1615 agt gat aat tta tgt aga caa act gaa aat ct #c aaa aca tca aaa agt     5092 Ser Asp Asn Leu Cys Arg Gln Thr Glu Asn Le #u Lys Thr Ser Lys Ser 1620                1625 #                1630 atc ttt ttg aaa gtt aaa gta cat gaa aat gt #a gaa aaa gaa aca gca     5140 Ile Phe Leu Lys Val Lys Val His Glu Asn Va #l Glu Lys Glu Thr Ala 1635                1640 #                1645 aaa agt cct gca act tgt tac aca aat cag tc #c cct tat tca gtc att     5188 Lys Ser Pro Ala Thr Cys Tyr Thr Asn Gln Se #r Pro Tyr Ser Val Ile 1650                1655 #                1660 gaa aat tca gcc tta gct ttt tac aca agt tg #t agt aga aaa act tct     5236 Glu Asn Ser Ala Leu Ala Phe Tyr Thr Ser Cy #s Ser Arg Lys Thr Ser 1665                1670 #                1675  #               1680 gtg agt cag act tca tta ctt gaa gca aaa aa #a tgg ctt aga gaa gga     5284 Val Ser Gln Thr Ser Leu Leu Glu Ala Lys Ly #s Trp Leu Arg Glu Gly 1685                1690 #                1695 ata ttt gat ggt caa cca gaa aga ata aat ac #t gca gat tat gta gga     5332 Ile Phe Asp Gly Gln Pro Glu Arg Ile Asn Th #r Ala Asp Tyr Val Gly 1700                1705 #                1710 aat tat ttg tat gaa aat aat tca aac agt ac #t ata gct gaa aat gac     5380 Asn Tyr Leu Tyr Glu Asn Asn Ser Asn Ser Th #r Ile Ala Glu Asn Asp 1715                1720 #                1725 aaa aat cat ctc tcc gaa aaa caa gat act ta #t tta agt aac agt agc     5428 Lys Asn His Leu Ser Glu Lys Gln Asp Thr Ty #r Leu Ser Asn Ser Ser 1730                1735 #                1740 atg tct aac agc tat tcc tac cat tct gat ga #g gta tat aat gat tca     5476 Met Ser Asn Ser Tyr Ser Tyr His Ser Asp Gl #u Val Tyr Asn Asp Ser 1745                1750 #                1755  #               1760 gga tat ctc tca aaa aat aaa ctt gat tct gg #t att gag cca gta ttg     5524 Gly Tyr Leu Ser Lys Asn Lys Leu Asp Ser Gl #y Ile Glu Pro Val Leu 1765                1770 #                1775 aag aat gtt gaa gat caa aaa aac act agt tt #t tcc aaa gta ata tcc     5572 Lys Asn Val Glu Asp Gln Lys Asn Thr Ser Ph #e Ser Lys Val Ile Ser 1780                1785 #                1790 aat gta aaa gat gca aat gca tac cca caa ac #t gta aat gaa gat att     5620 Asn Val Lys Asp Ala Asn Ala Tyr Pro Gln Th #r Val Asn Glu Asp Ile 1795                1800 #                1805 tgc gtt gag gaa ctt gtg act agc tct tca cc #c tgc aaa aat aaa aat     5668 Cys Val Glu Glu Leu Val Thr Ser Ser Ser Pr #o Cys Lys Asn Lys Asn 1810                1815 #                1820 gca gcc att aaa ttg tcc ata tct aat agt aa #t aat ttt gag gta ggg     5716 Ala Ala Ile Lys Leu Ser Ile Ser Asn Ser As #n Asn Phe Glu Val Gly 1825                1830 #                1835  #               1840 cca cct gca ttt agg ata gcc agt ggt aaa at #c cgt ttg tgt tca cat     5764 Pro Pro Ala Phe Arg Ile Ala Ser Gly Lys Il #e Arg Leu Cys Ser His 1845                1850 #                1855 gaa aca att aaa aaa gtg aaa gac ata ttt ac #a gac agt ttc agc aaa     5812 Glu Thr Ile Lys Lys Val Lys Asp Ile Phe Th #r Asp Ser Phe Ser Lys 1860                1865 #                1870 gta att aag gaa aac aac gag aat aaa tca aa #a att tgc caa acg aaa     5860 Val Ile Lys Glu Asn Asn Glu Asn Lys Ser Ly #s Ile Cys Gln Thr Lys 1875                1880 #                1885 att atg gca ggt tgt tac gag gca ttg gat ga #t tca gag gat att ctt     5908 Ile Met Ala Gly Cys Tyr Glu Ala Leu Asp As #p Ser Glu Asp Ile Leu 1890                1895 #                1900 cat aac tct cta gat aat gat gaa tgt agc at #g cat tca cat aag gtt     5956 His Asn Ser Leu Asp Asn Asp Glu Cys Ser Me #t His Ser His Lys Val 1905                1910 #                1915  #               1920 ttt gct gac att cag agt gaa gaa att tta ca #a cat aac caa aat atg     6004 Phe Ala Asp Ile Gln Ser Glu Glu Ile Leu Gl #n His Asn Gln Asn Met 1925                1930 #                1935 tct gga ttg gag aaa gtt tct aaa ata tca cc #t tgt gat gtt agt ttg     6052 Ser Gly Leu Glu Lys Val Ser Lys Ile Ser Pr #o Cys Asp Val Ser Leu 1940                1945 #                1950 gaa act tca gat ata tgt aaa tgt agt ata gg #g aag ctt cat aag tca     6100 Glu Thr Ser Asp Ile Cys Lys Cys Ser Ile Gl #y Lys Leu His Lys Ser 1955                1960 #                1965 gtc tca tct gca aat act tgt ggg att ttt ag #c aca gca agt gga aaa     6148 Val Ser Ser Ala Asn Thr Cys Gly Ile Phe Se #r Thr Ala Ser Gly Lys 1970                1975 #                1980 tct gtc cag gta tca gat gct tca tta caa aa #c gca aga caa gtg ttt     6196 Ser Val Gln Val Ser Asp Ala Ser Leu Gln As #n Ala Arg Gln Val Phe 1985                1990 #                1995  #               2000 tct gaa ata gaa gat agt acc aag caa gtc tt #t tcc aaa gta ttg ttt     6244 Ser Glu Ile Glu Asp Ser Thr Lys Gln Val Ph #e Ser Lys Val Leu Phe 2005                2010 #                2015 aaa agt aac gaa cat tca gac cag ctc aca ag #a gaa gaa aat act gct     6292 Lys Ser Asn Glu His Ser Asp Gln Leu Thr Ar #g Glu Glu Asn Thr Ala 2020                2025 #                2030 ata cgt act cca gaa cat tta ata tcc caa aa #a ggc ttt tca tat aat     6340 Ile Arg Thr Pro Glu His Leu Ile Ser Gln Ly #s Gly Phe Ser Tyr Asn 2035                2040 #                2045 gtg gta aat tca tct gct ttc tct gga ttt ag #t aca gca agt gga aag     6388 Val Val Asn Ser Ser Ala Phe Ser Gly Phe Se #r Thr Ala Ser Gly Lys 2050                2055 #                2060 caa gtt tcc att tta gaa agt tcc tta cac aa #a gtt aag gga gtg tta     6436 Gln Val Ser Ile Leu Glu Ser Ser Leu His Ly #s Val Lys Gly Val Leu 2065                2070 #                2075  #               2080 gag gaa ttt gat tta atc aga act gag cat ag #t ctt cac tat tca cct     6484 Glu Glu Phe Asp Leu Ile Arg Thr Glu His Se #r Leu His Tyr Ser Pro 2085                2090 #                2095 acg tct aga caa aat gta tca aaa ata ctt cc #t cgt gtt gat aag aga     6532 Thr Ser Arg Gln Asn Val Ser Lys Ile Leu Pr #o Arg Val Asp Lys Arg 2100                2105 #                2110 aac cca gag cac tgt gta aac tca gaa atg ga #a aaa acc tgc agt aaa     6580 Asn Pro Glu His Cys Val Asn Ser Glu Met Gl #u Lys Thr Cys Ser Lys 2115                2120 #                2125 gaa ttt aaa tta tca aat aac tta aat gtt ga #a ggt ggt tct tca gaa     6628 Glu Phe Lys Leu Ser Asn Asn Leu Asn Val Gl #u Gly Gly Ser Ser Glu 2130                2135 #                2140 aat aat cac tct att aaa gtt tct cca tat ct #c tct caa ttt caa caa     6676 Asn Asn His Ser Ile Lys Val Ser Pro Tyr Le #u Ser Gln Phe Gln Gln 2145                2150 #                2155  #               2160 gac aaa caa cag ttg gta tta gga acc aaa gt #c tca ctt gtt gag aac     6724 Asp Lys Gln Gln Leu Val Leu Gly Thr Lys Va #l Ser Leu Val Glu Asn 2165                2170 #                2175 att cat gtt ttg gga aaa gaa cag gct tca cc #t aaa aac gta aaa atg     6772 Ile His Val Leu Gly Lys Glu Gln Ala Ser Pr #o Lys Asn Val Lys Met 2180                2185 #                2190 gaa att ggt aaa act gaa act ttt tct gat gt #t cct gtg aaa aca aat     6820 Glu Ile Gly Lys Thr Glu Thr Phe Ser Asp Va #l Pro Val Lys Thr Asn 2195                2200 #                2205 ata gaa gtt tgt tct act tac tcc aaa gat tc #a gaa aac tac ttt gaa     6868 Ile Glu Val Cys Ser Thr Tyr Ser Lys Asp Se #r Glu Asn Tyr Phe Glu 2210                2215 #                2220 aca gaa gca gta gaa att gct aaa gct ttt at #g gaa gat gat gaa ctg     6916 Thr Glu Ala Val Glu Ile Ala Lys Ala Phe Me #t Glu Asp Asp Glu Leu 2225                2230 #                2235  #               2240 aca gat tct aaa ctg cca agt cat gcc aca ca #t tct ctt ttt aca tgt     6964 Thr Asp Ser Lys Leu Pro Ser His Ala Thr Hi #s Ser Leu Phe Thr Cys 2245                2250 #                2255 ccc gaa aat gag gaa atg gtt ttg tca aat tc #a aga att gga aaa aga     7012 Pro Glu Asn Glu Glu Met Val Leu Ser Asn Se #r Arg Ile Gly Lys Arg 2260                2265 #                2270 aga gga gag ccc ctt atc tta gtg gga gaa cc #c tca atc aaa aga aac     7060 Arg Gly Glu Pro Leu Ile Leu Val Gly Glu Pr #o Ser Ile Lys Arg Asn 2275                2280 #                2285 tta tta aat gaa ttt gac agg ata ata gaa aa #t caa gaa aaa tcc tta     7108 Leu Leu Asn Glu Phe Asp Arg Ile Ile Glu As #n Gln Glu Lys Ser Leu 2290                2295 #                2300 aag gct tca aaa agc act cca gat ggc aca at #a aaa gat cga aga ttg     7156 Lys Ala Ser Lys Ser Thr Pro Asp Gly Thr Il #e Lys Asp Arg Arg Leu 2305                2310 #                2315  #               2320 ttt atg cat cat gtt tct tta gag ccg att ac #c tgt gta ccc ttt cgc     7204 Phe Met His His Val Ser Leu Glu Pro Ile Th #r Cys Val Pro Phe Arg 2325                2330 #                2335 aca act aag gaa cgt caa gag ata cag aat cc #a aat ttt acc gca cct     7252 Thr Thr Lys Glu Arg Gln Glu Ile Gln Asn Pr #o Asn Phe Thr Ala Pro 2340                2345 #                2350 ggt caa gaa ttt ctg tct aaa tct cat ttg ta #t gaa cat ctg act ttg     7300 Gly Gln Glu Phe Leu Ser Lys Ser His Leu Ty #r Glu His Leu Thr Leu 2355                2360 #                2365 gaa aaa tct tca agc aat tta gca gtt tca gg #a cat cca ttt tat caa     7348 Glu Lys Ser Ser Ser Asn Leu Ala Val Ser Gl #y His Pro Phe Tyr Gln 2370                2375 #                2380 gtt tct gct aca aga aat gaa aaa atg aga ca #c ttg att act aca ggc     7396 Val Ser Ala Thr Arg Asn Glu Lys Met Arg Hi #s Leu Ile Thr Thr Gly 2385                2390 #                2395  #               2400 aga cca acc aaa gtc ttt gtt cca cct ttt aa #a act aaa tca cat ttt     7444 Arg Pro Thr Lys Val Phe Val Pro Pro Phe Ly #s Thr Lys Ser His Phe 2405                2410 #                2415 cac aga gtt gaa cag tgt gtt agg aat att aa #c ttg gag gaa aac aga     7492 His Arg Val Glu Gln Cys Val Arg Asn Ile As #n Leu Glu Glu Asn Arg 2420                2425 #                2430 caa aag caa aac att gat gga cat ggc tct ga #t gat agt aaa aat aag     7540 Gln Lys Gln Asn Ile Asp Gly His Gly Ser As #p Asp Ser Lys Asn Lys 2435                2440 #                2445 att aat gac aat gag att cat cag ttt aac aa #a aac aac tcc aat caa     7588 Ile Asn Asp Asn Glu Ile His Gln Phe Asn Ly #s Asn Asn Ser Asn Gln 2450                2455 #                2460 gca gca gct gta act ttc aca aag tgt gaa ga #a gaa cct tta gat tta     7636 Ala Ala Ala Val Thr Phe Thr Lys Cys Glu Gl #u Glu Pro Leu Asp Leu 2465                2470 #                2475  #               2480 att aca agt ctt cag aat gcc aga gat ata ca #g gat atg cga att aag     7684 Ile Thr Ser Leu Gln Asn Ala Arg Asp Ile Gl #n Asp Met Arg Ile Lys 2485                2490 #                2495 aag aaa caa agg caa cgc gtc ttt cca cag cc #a ggc agt ctg tat ctt     7732 Lys Lys Gln Arg Gln Arg Val Phe Pro Gln Pr #o Gly Ser Leu Tyr Leu 2500                2505 #                2510 gca aaa aca tcc act ctg cct cga atc tct ct #g aaa gca gca gta gga     7780 Ala Lys Thr Ser Thr Leu Pro Arg Ile Ser Le #u Lys Ala Ala Val Gly 2515                2520 #                2525 ggc caa gtt ccc tct gcg tgt tct cat aaa ca #g ctg tat acg tat ggc     7828 Gly Gln Val Pro Ser Ala Cys Ser His Lys Gl #n Leu Tyr Thr Tyr Gly 2530                2535 #                2540 gtt tct aaa cat tgc ata aaa att aac agc aa #a aat gca gag tct ttt     7876 Val Ser Lys His Cys Ile Lys Ile Asn Ser Ly #s Asn Ala Glu Ser Phe 2545                2550 #                2555  #               2560 cag ttt cac act gaa gat tat ttt ggt aag ga #a agt tta tgg act gga     7924 Gln Phe His Thr Glu Asp Tyr Phe Gly Lys Gl #u Ser Leu Trp Thr Gly 2565                2570 #                2575 aaa gga ata cag ttg gct gat ggt gga tgg ct #c ata ccc tcc aat gat     7972 Lys Gly Ile Gln Leu Ala Asp Gly Gly Trp Le #u Ile Pro Ser Asn Asp 2580                2585 #                2590 gga aag gct gga aaa gaa gaa ttt tat agg gc #t ctg tgt gac act cca     8020 Gly Lys Ala Gly Lys Glu Glu Phe Tyr Arg Al #a Leu Cys Asp Thr Pro 2595                2600 #                2605 ggt gtg gat cca aag ctt att tct aga att tg #g gtt tat aat cac tat     8068 Gly Val Asp Pro Lys Leu Ile Ser Arg Ile Tr #p Val Tyr Asn His Tyr 2610                2615 #                2620 aga tgg atc ata tgg aaa ctg gca gct atg ga #a tgt gcc ttt cct aag     8116 Arg Trp Ile Ile Trp Lys Leu Ala Ala Met Gl #u Cys Ala Phe Pro Lys 2625                2630 #                2635  #               2640 gaa ttt gct aat aga tgc cta agc cca gaa ag #g gtg ctt ctt caa cta     8164 Glu Phe Ala Asn Arg Cys Leu Ser Pro Glu Ar #g Val Leu Leu Gln Leu 2645                2650 #                2655 aaa tac aga tat gat acg gaa att gat aga ag #c aga aga tcg gct ata     8212 Lys Tyr Arg Tyr Asp Thr Glu Ile Asp Arg Se #r Arg Arg Ser Ala Ile 2660                2665 #                2670 aaa aag ata atg gaa agg gat gac aca gct gc #a aaa aca ctt gtt ctc     8260 Lys Lys Ile Met Glu Arg Asp Asp Thr Ala Al #a Lys Thr Leu Val Leu 2675                2680 #                2685 tgt gtt tct gac ata att tca ttg agc gca aa #t ata tct gaa act tct     8308 Cys Val Ser Asp Ile Ile Ser Leu Ser Ala As #n Ile Ser Glu Thr Ser 2690                2695 #                2700 agc aat aaa act agt agt gca gat acc caa aa #a gtg gcc att att gaa     8356 Ser Asn Lys Thr Ser Ser Ala Asp Thr Gln Ly #s Val Ala Ile Ile Glu 2705                2710 #                2715  #               2720 ctt aca gat ggg tgg tat gct gtt aag gcc ca #g tta gat cct ccc ctc     8404 Leu Thr Asp Gly Trp Tyr Ala Val Lys Ala Gl #n Leu Asp Pro Pro Leu 2725                2730 #                2735 tta gct gtc tta aag aat ggc aga ctg aca gt #t ggt cag aag att att     8452 Leu Ala Val Leu Lys Asn Gly Arg Leu Thr Va #l Gly Gln Lys Ile Ile 2740                2745 #                2750 ctt cat gga gca gaa ctg gtg ggc tct cct ga #t gcc tgt aca cct ctt     8500 Leu His Gly Ala Glu Leu Val Gly Ser Pro As #p Ala Cys Thr Pro Leu 2755                2760 #                2765 gaa gcc cca gaa tct ctt atg tta aag att tc #t gct aac agt act cgg     8548 Glu Ala Pro Glu Ser Leu Met Leu Lys Ile Se #r Ala Asn Ser Thr Arg 2770                2775 #                2780 cct gct cgc tgg tat acc aaa ctt gga ttc tt #t cct gac cct aga cct     8596 Pro Ala Arg Trp Tyr Thr Lys Leu Gly Phe Ph #e Pro Asp Pro Arg Pro 2785                2790 #                2795  #               2800 ttt cct ctg ccc tta tca tcg ctt ttc agt ga #t gga gga aat gtt ggt     8644 Phe Pro Leu Pro Leu Ser Ser Leu Phe Ser As #p Gly Gly Asn Val Gly 2805                2810 #                2815 tgt gtt gat gta att att caa aga gca tac cc #t ata cag cgg atg gag     8692 Cys Val Asp Val Ile Ile Gln Arg Ala Tyr Pr #o Ile Gln Arg Met Glu 2820                2825 #                2830 aag aca tca tct gga tta tac ata ttt cgc aa #t gaa aga gag gaa gaa     8740 Lys Thr Ser Ser Gly Leu Tyr Ile Phe Arg As #n Glu Arg Glu Glu Glu 2835                2840 #                2845 aag gaa gca gca aaa tat gtg gag gcc caa ca #a aag aga cta gaa gcc     8788 Lys Glu Ala Ala Lys Tyr Val Glu Ala Gln Gl #n Lys Arg Leu Glu Ala 2850                2855 #                2860 tta ttc act aaa att cag gag gaa ttt gaa ga #a cat gaa gaa aac aca     8836 Leu Phe Thr Lys Ile Gln Glu Glu Phe Glu Gl #u His Glu Glu Asn Thr 2865                2870 #                2875  #               2880 aca aaa cca tat tta cca tca cgt gca cta ac #a aga cag caa gtt cgt     8884 Thr Lys Pro Tyr Leu Pro Ser Arg Ala Leu Th #r Arg Gln Gln Val Arg 2885                2890 #                2895 gct ttg caa gat ggt gca gag ctt tat gaa gc #a gtg aag aat gca gca     8932 Ala Leu Gln Asp Gly Ala Glu Leu Tyr Glu Al #a Val Lys Asn Ala Ala 2900                2905 #                2910 gac cca gct tac ctt gag ggt tat ttc agt ga #a gag cag tta aga gcc     8980 Asp Pro Ala Tyr Leu Glu Gly Tyr Phe Ser Gl #u Glu Gln Leu Arg Ala 2915                2920 #                2925 ttg aat aat cac agg caa atg ttg aat gat aa #g aaa caa gct cag atc     9028 Leu Asn Asn His Arg Gln Met Leu Asn Asp Ly #s Lys Gln Ala Gln Ile 2930                2935 #                2940 cag ttg gaa att agg aag gcc atg gaa tct gc #t gaa caa aag gaa caa     9076 Gln Leu Glu Ile Arg Lys Ala Met Glu Ser Al #a Glu Gln Lys Glu Gln 2945                2950 #                2955  #               2960 ggt tta tca agg gat gtc aca acc gtg tgg aa #g ttg cgt att gta agc     9124 Gly Leu Ser Arg Asp Val Thr Thr Val Trp Ly #s Leu Arg Ile Val Ser 2965                2970 #                2975 tat tca aaa aaa gaa aaa gat tca gtt ata ct #g agt att tgg cgt cca     9172 Tyr Ser Lys Lys Glu Lys Asp Ser Val Ile Le #u Ser Ile Trp Arg Pro 2980                2985 #                2990 tca tca gat tta tat tct ctg tta aca gaa gg #a aag aga tac aga att     9220 Ser Ser Asp Leu Tyr Ser Leu Leu Thr Glu Gl #y Lys Arg Tyr Arg Ile 2995                3000 #                3005 tat cat ctt gca act tca aaa tct aaa agt aa #a tct gaa aga gct aac     9268 Tyr His Leu Ala Thr Ser Lys Ser Lys Ser Ly #s Ser Glu Arg Ala Asn 3010                3015 #                3020 ata cag tta gca gcg aca aaa aaa act cag ta #t caa caa cta ccg gtt     9316 Ile Gln Leu Ala Ala Thr Lys Lys Thr Gln Ty #r Gln Gln Leu Pro Val 3025                3030 #                3035  #               3040 tca gat gaa att tta ttt cag att tac cag cc #a cgg gag ccc ctt cac     9364 Ser Asp Glu Ile Leu Phe Gln Ile Tyr Gln Pr #o Arg Glu Pro Leu His 3045                3050 #                3055 ttc agc aaa ttt tta gat cca gac ttt cag cc #a tct tgt tct gag gtg     9412 Phe Ser Lys Phe Leu Asp Pro Asp Phe Gln Pr #o Ser Cys Ser Glu Val 3060                3065 #                3070 gac cta ata gga ttt gtc gtt tct gtt gtg aa #a aaa aca gga ctt gcc     9460 Asp Leu Ile Gly Phe Val Val Ser Val Val Ly #s Lys Thr Gly Leu Ala 3075                3080 #                3085 cct ttc gtc tat ttg tca gac gaa tgt tac aa #t tta ctg gca ata aag     9508 Pro Phe Val Tyr Leu Ser Asp Glu Cys Tyr As #n Leu Leu Ala Ile Lys 3090                3095 #                3100 ttt tgg ata gac ctt aat gag gac att att aa #g cct cat atg tta att     9556 Phe Trp Ile Asp Leu Asn Glu Asp Ile Ile Ly #s Pro His Met Leu Ile 3105                3110 #                3115  #               3120 gct gca agc aac ctc cag tgg cga cca gaa tc #c aaa tca ggc ctt ctt     9604 Ala Ala Ser Asn Leu Gln Trp Arg Pro Glu Se #r Lys Ser Gly Leu Leu 3125                3130 #                3135 act tta ttt gct gga gat ttt tct gtg ttt tc #t gct agt cca aaa gag     9652 Thr Leu Phe Ala Gly Asp Phe Ser Val Phe Se #r Ala Ser Pro Lys Glu 3140                3145 #                3150 ggc cac ttt caa gag aca ttc aac aaa atg aa #a aat act gtt gag aat     9700 Gly His Phe Gln Glu Thr Phe Asn Lys Met Ly #s Asn Thr Val Glu Asn 3155                3160 #                3165 att gac ata ctt tgc aat gaa gca gaa aac aa #g ctt atg cat ata ctg     9748 Ile Asp Ile Leu Cys Asn Glu Ala Glu Asn Ly #s Leu Met His Ile Leu 3170                3175 #                3180 cat gca aat gat ccc aag tgg tcc acc cca ac #t aaa gac tgt act tca     9796 His Ala Asn Asp Pro Lys Trp Ser Thr Pro Th #r Lys Asp Cys Thr Ser 3185                3190 #                 319 #5               3200 ggg ccg tac act gct caa atc att cct ggt ac #a gga aac aag ctt ctg     9844 Gly Pro Tyr Thr Ala Gln Ile Ile Pro Gly Th #r Gly Asn Lys Leu Leu 3205                3210 #                3215 atg tct tct cct aat tgt gag ata tat tat ca #a agt cct tta tca ctt     9892 Met Ser Ser Pro Asn Cys Glu Ile Tyr Tyr Gl #n Ser Pro Leu Ser Leu 3220                3225 #                3230 tgt atg gcc aaa agg aag tct gtt tcc aca cc #t gtc tca gcc cag atg     9940 Cys Met Ala Lys Arg Lys Ser Val Ser Thr Pr #o Val Ser Ala Gln Met 3235                3240 #                3245 act tca aag tct tgt aaa ggg gag aaa gag at #t gat gac caa aag aac     9988 Thr Ser Lys Ser Cys Lys Gly Glu Lys Glu Il #e Asp Asp Gln Lys Asn 3250                3255 #                3260 tgc aaa aag aga aga gcc ttg gat ttc ttg ag #t aga ctg cct tta cct    10036 Cys Lys Lys Arg Arg Ala Leu Asp Phe Leu Se #r Arg Leu Pro Leu Pro 3265                3270 #                3275  #               3280 cca cct gtt agt ccc att tgt aca ttt gtt tc #t ccg gct gca cag aag    10084 Pro Pro Val Ser Pro Ile Cys Thr Phe Val Se #r Pro Ala Ala Gln Lys 3285                3290 #                3295 gca ttt cag cca cca agg agt tgt ggc acc aa #a tac gaa aca ccc ata    10132 Ala Phe Gln Pro Pro Arg Ser Cys Gly Thr Ly #s Tyr Glu Thr Pro Ile 3300                3305 #                3310 aag aaa aaa gaa ctg aat tct cct cag atg ac #t cca ttt aaa aaa ttc    10180 Lys Lys Lys Glu Leu Asn Ser Pro Gln Met Th #r Pro Phe Lys Lys Phe 3315                3320 #                3325 aat gaa att tct ctt ttg gaa agt aat tca at #a gct gac gaa gaa ctt    10228 Asn Glu Ile Ser Leu Leu Glu Ser Asn Ser Il #e Ala Asp Glu Glu Leu 3330                3335 #                3340 gca ttg ata aat acc caa gct ctt ttg tct gg #t tca aca gga gaa aaa    10276 Ala Leu Ile Asn Thr Gln Ala Leu Leu Ser Gl #y Ser Thr Gly Glu Lys 3345                3350 #                3355  #               3360 caa ttt ata tct gtc agt gaa tcc act agg ac #t gct ccc acc agt tca    10324 Gln Phe Ile Ser Val Ser Glu Ser Thr Arg Th #r Ala Pro Thr Ser Ser 3365                3370 #                3375 gaa gat tat ctc aga ctg aaa cga cgt tgt ac #t aca tct ctg atc aaa    10372 Glu Asp Tyr Leu Arg Leu Lys Arg Arg Cys Th #r Thr Ser Leu Ile Lys 3380                3385 #                3390 gaa cag gag agt tcc cag gcc agt acg gaa ga #a tgt gag aaa aat aag    10420 Glu Gln Glu Ser Ser Gln Ala Ser Thr Glu Gl #u Cys Glu Lys Asn Lys 3395                3400 #                3405 cag gac aca att aca act aaa aaa tat atc ta #agcatttg caaaggcgac      10470 Gln Asp Thr Ile Thr Thr Lys Lys Tyr Ile 3410                3415 aataaattat tgacgcttaa cctttccagt ttataagact ggaatataat tt #caaaccac  10530 acattagtac ttatgttgcm caatgagaaa agaaattagt ttcaaattta cc #tcagcgtt  10590 tgtgtatcgg gcaaaaatcg ttttgcccga ttccgtattg gtatactttt gc #ctcagttg  10650 catatcctaa aactaaatgt aatttattaa ctaatcaaga aaaacatctt tg #gctgagct  10710 cggtggctca tgcctgtaat cccaacactt tgagaagctg aggtgggagg ag #tgcttgag  10770 gccaggagtt caagaccagc ctgggcaaca tagggagacc ccatctttac ga #agaaaaaa  10830 aaaaagggga aaagaaaatc ttttaaatct ttggatttca ctacaagtat ta #ttttacaa  10890 gtgaaataaa cataccattt tcttttagat tgtgtcatta aatggaatga gg #tctcttag  10950 tacagttatt ttgatgcaga taattccttt tagtttagct actattttag gg #gatttttt  11010 ttagaggtaa ctcactatga aatagttccc cttaatgcaa atatgttggt tc #tgcaatag  11070 ttccatcctg ttcaaaartc rggrtgaawa tgaagagtgg tgttyccttt tg #agcaattc  11130 tcatccttaa gtcagcrtga ttataagaaa aatagaaccc ycagtgtaac yc #taattcct  11190 ttttrctatt ccagtgtgat ctctgaaakt aaattacttc mactaaaaat tc #aaaaactt  11250 waamtcagaa rawttcawag twgatttatt ttt        #                   #      11283 (2) INFORMATION FOR SEQ ID NO:4:      (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH:  3418           (B) TYPE: amino acid           (C) STRANDEDNESS: single           (D) TOPOLOGY: unknown     (ix) FEATURE:           (A) NAME/KEY: BRCA2 pro #tein      (x) PUBLICATION INFORMATION:           (A) AUTHORS:  Wooster,  #R. et al.           (B) TITLE:  Identificat #ion of the breast cancer                susceptabili #ty gene BRCA2           (C) JOURNAL:  Nature           (D) VOLUME:  379           (E) PAGES:  789-792           (F) DATE:  1995           (K) RELEVANT RESIDUES I #N SEQ ID NO:4:  granin box domain at                amino aci #ds 3334-3344     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: #4: Met Pro Ile Gly Ser Lys Glu Arg Pro Thr Ph #e Phe Glu Ile Phe Lys 1               5    #                10   #                15 Thr Arg Cys Asn Lys Ala Asp Leu Gly Pro Il #e Ser Leu Asn Trp Phe             20       #            25       #            30 Glu Glu Leu Ser Ser Glu Ala Pro Pro Tyr As #n Ser Glu Pro Ala Glu         35           #        40           #        45 Glu Ser Glu His Lys Asn Asn Asn Tyr Glu Pr #o Asn Leu Phe Lys Thr     50               #    55               #    60 Pro Gln Arg Lys Pro Ser Tyr Asn Gln Leu Al #a Ser Thr Pro Ile Ile 65                   #70                   #75                   #80 Phe Lys Glu Gln Gly Leu Thr Leu Pro Leu Ty #r Gln Ser Pro Val Lys                 85   #                90   #                95 Glu Leu Asp Lys Phe Lys Leu Asp Leu Gly Ar #g Asn Val Pro Asn Ser             100       #           105       #           110 Arg His Lys Ser Leu Arg Thr Val Lys Tyr Ly #s Met Asp Gln Ala Asp         115           #       120           #       125 Asp Val Ser Cys Pro Leu Leu Asn Ser Cys Le #u Ser Glu Ser Pro Val     130               #   135               #   140 Val Leu Gln Cys Thr His Val Thr Pro Gln Ar #g Asp Lys Ser Val Val 145                 1 #50                 1 #55                 1 #60 Cys Gly Ser Leu Phe His Thr Pro Lys Phe Va #l Lys Gly Arg Gln Thr                 165   #               170   #               175 Pro Lys His Ile Ser Glu Ser Leu Gly Ala Gl #u Val Asp Pro Asp Met             180       #           185       #           190 Ser Trp Ser Ser Ser Leu Ala Thr Pro Pro Th #r Leu Ser Ser Thr Val         195           #       200           #       205 Leu Ile Val Arg Asn Glu Glu Ala Ser Glu Th #r Val Phe Pro His Asp     210               #   215               #   220 Thr Thr Ala Asn Val Lys Ser Tyr Phe Ser As #n His Asp Glu Ser Leu 225                 2 #30                 2 #35                 2 #40 Lys Lys Asn Asp Arg Phe Ile Ala Ser Val Th #r Asp Ser Glu Asn Thr                 245   #               250   #               255 Asn Gln Arg Glu Ala Ala Ser His Gly Phe Gl #y Lys Thr Ser Gly Asn             260       #           265       #           270 Ser Phe Lys Val Asn Ser Cys Lys Asp His Il #e Gly Lys Ser Met Pro         275           #       280           #       285 Asn Val Leu Glu Asp Glu Val Tyr Glu Thr Va #l Val Asp Thr Ser Glu     290               #   295               #   300 Glu Asp Ser Phe Ser Leu Cys Phe Ser Lys Cy #s Arg Thr Lys Asn Leu 305                 3 #10                 3 #15                 3 #20 Gln Lys Val Arg Thr Ser Lys Thr Arg Lys Ly #s Ile Phe His Glu Ala                 325   #               330   #               335 Asn Ala Asp Glu Cys Glu Lys Ser Lys Asn Gl #n Val Lys Glu Lys Tyr             340       #           345       #           350 Ser Phe Val Ser Glu Val Glu Pro Asn Asp Th #r Asp Pro Leu Asp Ser         355           #       360           #       365 Asn Val Ala His Gln Lys Pro Phe Glu Ser Gl #y Ser Asp Lys Ile Ser     370               #   375               #   380 Lys Glu Val Val Pro Ser Leu Ala Cys Glu Tr #p Ser Gln Leu Thr Leu 385                 3 #90                 3 #95                 4 #00 Ser Gly Leu Asn Gly Ala Gln Met Glu Lys Il #e Pro Leu Leu His Ile                 405   #               410   #               415 Ser Ser Cys Asp Gln Asn Ile Ser Glu Lys As #p Leu Leu Asp Thr Glu             420       #           425       #           430 Asn Lys Arg Lys Lys Asp Phe Leu Thr Ser Gl #u Asn Ser Leu Pro Arg         435           #       440           #       445 Ile Ser Ser Leu Pro Lys Ser Glu Lys Pro Le #u Asn Glu Glu Thr Val     450               #   455               #   460 Val Asn Lys Arg Asp Glu Glu Gln His Leu Gl #u Ser His Thr Asp Cys 465                 4 #70                 4 #75                 4 #80 Ile Leu Ala Val Lys Gln Ala Ile Ser Gly Th #r Ser Pro Val Ala Ser                 485   #               490   #               495 Ser Phe Gln Gly Ile Lys Lys Ser Ile Phe Ar #g Ile Arg Glu Ser Pro             500       #           505       #           510 Lys Glu Thr Phe Asn Ala Ser Phe Ser Gly Hi #s Met Thr Asp Pro Asn         515           #       520           #       525 Phe Lys Lys Glu Thr Glu Ala Ser Glu Ser Gl #y Leu Glu Ile His Thr     530               #   535               #   540 Val Cys Ser Gln Lys Glu Asp Ser Leu Cys Pr #o Asn Leu Ile Asp Asn 545                 5 #50                 5 #55                 5 #60 Gly Ser Trp Pro Ala Thr Thr Thr Gln Asn Se #r Val Ala Leu Lys Asn                 565   #               570   #               575 Ala Gly Leu Ile Ser Thr Leu Lys Lys Lys Th #r Asn Lys Phe Ile Tyr             580       #           585       #           590 Ala Ile His Asp Glu Thr Phe Tyr Lys Gly Ly #s Lys Ile Pro Lys Asp         595           #       600           #       605 Gln Lys Ser Glu Leu Ile Asn Cys Ser Ala Gl #n Phe Glu Ala Asn Ala     610               #   615               #   620 Phe Glu Ala Pro Leu Thr Phe Ala Asn Ala As #p Ser Gly Leu Leu His 625                 6 #30                 6 #35                 6 #40 Ser Ser Val Lys Arg Ser Cys Ser Gln Asn As #p Ser Glu Glu Pro Thr                 645   #               650   #               655 Leu Ser Leu Thr Ser Ser Phe Gly Thr Ile Le #u Arg Lys Cys Ser Arg             660       #           665       #           670 Asn Glu Thr Cys Ser Asn Asn Thr Val Ile Se #r Gln Asp Leu Asp Tyr         675           #       680           #       685 Lys Glu Ala Lys Cys Asn Lys Glu Lys Leu Gl #n Leu Phe Ile Thr Pro     690               #   695               #   700 Glu Ala Asp Ser Leu Ser Cys Leu Gln Glu Gl #y Gln Cys Glu Asn Asp 705                 7 #10                 7 #15                 7 #20 Pro Lys Ser Lys Lys Val Ser Asp Ile Lys Gl #u Glu Val Leu Ala Ala                 725   #               730   #               735 Ala Cys His Pro Val Gln His Ser Lys Val Gl #u Tyr Ser Asp Thr Asp             740       #           745       #           750 Phe Gln Ser Gln Lys Ser Leu Leu Tyr Asp Hi #s Glu Asn Ala Ser Thr         755           #       760           #       765 Leu Ile Leu Thr Pro Thr Ser Lys Asp Val Le #u Ser Asn Leu Val Met     770               #   775               #   780 Ile Ser Arg Gly Lys Glu Ser Tyr Lys Met Se #r Asp Lys Leu Lys Gly 785                 7 #90                 7 #95                 8 #00 Asn Asn Tyr Glu Ser Asp Val Glu Leu Thr Ly #s Asn Ile Pro Met Glu                 805   #               810   #               815 Lys Asn Gln Asp Val Cys Ala Leu Asn Glu As #n Tyr Lys Asn Val Glu             820       #           825       #           830 Leu Leu Pro Pro Glu Lys Tyr Met Arg Val Al #a Ser Pro Ser Arg Lys         835           #       840           #       845 Val Gln Phe Asn Gln Asn Thr Asn Leu Arg Va #l Ile Gln Lys Asn Gln     850               #   855               #   860 Glu Glu Thr Thr Ser Ile Ser Lys Ile Thr Va #l Asn Pro Asp Ser Glu 865                 8 #70                 8 #75                 8 #80 Glu Leu Phe Ser Asp Asn Glu Asn Asn Phe Va #l Phe Gln Val Ala Asn                 885   #               890   #               895 Glu Arg Asn Asn Leu Ala Leu Gly Asn Thr Ly #s Glu Leu His Glu Thr             900       #           905       #           910 Asp Leu Thr Cys Val Asn Glu Pro Ile Phe Ly #s Asn Ser Thr Met Val         915           #       920           #       925 Leu Tyr Gly Asp Thr Gly Asp Lys Gln Ala Th #r Gln Val Ser Ile Lys     930               #   935               #   940 Lys Asp Leu Val Tyr Val Leu Ala Glu Glu As #n Lys Asn Ser Val Lys 945                 9 #50                 9 #55                 9 #60 Gln His Ile Lys Met Thr Leu Gly Gln Asp Le #u Lys Ser Asp Ile Ser                 965   #               970   #               975 Leu Asn Ile Asp Lys Ile Pro Glu Lys Asn As #n Asp Tyr Met Asn Lys             980       #           985       #           990 Trp Ala Gly Leu Leu Gly Pro Ile Ser Asn Hi #s Ser Phe Gly Gly Ser         995           #       1000           #      1005 Phe Arg Thr Ala Ser Asn Lys Glu Ile Lys Le #u Ser Glu His Asn Ile     1010              #   1015               #  1020 Lys Lys Ser Lys Met Phe Phe Lys Asp Ile Gl #u Glu Gln Tyr Pro Thr 1025                1030 #                1035  #               1040 Ser Leu Ala Cys Val Glu Ile Val Asn Thr Le #u Ala Leu Asp Asn Gln                 1045  #               1050   #              1055 Lys Lys Leu Ser Lys Pro Gln Ser Ile Asn Th #r Val Ser Ala His Leu             1060      #           1065       #          1070 Gln Ser Ser Val Val Val Ser Asp Cys Lys As #n Ser His Ile Thr Pro         1075          #       1080           #      1085 Gln Met Leu Phe Ser Lys Gln Asp Phe Asn Se #r Asn His Asn Leu Thr     1090              #   1095               #  1100 Pro Ser Gln Lys Ala Glu Ile Thr Glu Leu Se #r Thr Ile Leu Glu Glu 1105                1110 #                1115  #               1120 Ser Gly Ser Gln Phe Glu Phe Thr Gln Phe Ar #g Lys Pro Ser Tyr Ile                 1125  #               1130   #              1135 Leu Gln Lys Ser Thr Phe Glu Val Pro Glu As #n Gln Met Thr Ile Leu             1140      #           1145       #          1150 Lys Thr Thr Ser Glu Glu Cys Arg Asp Ala As #p Leu His Val Ile Met         1155          #       1160           #      1165 Asn Ala Pro Ser Ile Gly Gln Val Asp Ser Se #r Lys Gln Phe Glu Gly     1170              #   1175               #  1180 Thr Val Glu Ile Lys Arg Lys Phe Ala Gly Le #u Leu Lys Asn Asp Cys 1185                1190 #                1195  #               1200 Asn Lys Ser Ala Ser Gly Tyr Leu Thr Asp Gl #u Asn Glu Val Gly Phe                 1205  #               1210   #              1215 Arg Gly Phe Tyr Ser Ala His Gly Thr Lys Le #u Asn Val Ser Thr Glu             1220      #           1225       #          1230 Ala Leu Gln Lys Ala Val Lys Leu Phe Ser As #p Ile Glu Asn Ile Ser         1235          #       1240           #      1245 Glu Glu Thr Ser Ala Glu Val His Pro Ile Se #r Leu Ser Ser Ser Lys     1250              #   1255               #  1260 Cys His Asp Ser Val Val Ser Met Phe Lys Il #e Glu Asn His Asn Asp 1265                1270 #                1275  #               1280 Lys Thr Val Ser Glu Lys Asn Asn Lys Cys Gl #n Leu Ile Leu Gln Asn                 1285  #               1290   #              1295 Asn Ile Glu Met Thr Thr Gly Thr Phe Val Gl #u Glu Ile Thr Glu Asn             1300      #           1305       #          1310 Tyr Lys Arg Asn Thr Glu Asn Glu Asp Asn Ly #s Tyr Thr Ala Ala Ser         1315          #       1320           #      1325 Arg Asn Ser His Asn Leu Glu Phe Asp Gly Se #r Asp Ser Ser Lys Asn     1330              #   1335               #  1340 Asp Thr Val Cys Ile His Lys Asp Glu Thr As #p Leu Leu Phe Thr Asp 1345                1350 #                1355  #               1360 Gln His Asn Ile Cys Leu Lys Leu Ser Gly Gl #n Phe Met Lys Glu Gly                 1365  #               1370   #              1375 Asn Thr Gln Ile Lys Glu Asp Leu Ser Asp Le #u Thr Phe Leu Glu Val             1380      #           1385       #          1390 Ala Lys Ala Gln Glu Ala Cys His Gly Asn Th #r Ser Asn Lys Glu Gln         1395          #       1400           #      1405 Leu Thr Ala Thr Lys Thr Glu Gln Asn Ile Ly #s Asp Phe Glu Thr Ser     1410              #   1415               #  1420 Asp Thr Phe Phe Gln Thr Ala Ser Gly Lys As #n Ile Ser Val Ala Lys 1425                1430 #                1435  #               1440 Glu Leu Phe Asn Lys Ile Val Asn Phe Phe As #p Gln Lys Pro Glu Glu                 1445  #               1450   #              1455 Leu His Asn Phe Ser Leu Asn Ser Glu Leu Hi #s Ser Asp Ile Arg Lys             1460      #           1465       #          1470 Asn Lys Met Asp Ile Leu Ser Tyr Glu Glu Th #r Asp Ile Val Lys His         1475          #       1480           #      1485 Lys Ile Leu Lys Glu Ser Val Pro Val Gly Th #r Gly Asn Gln Leu Val     1490              #   1495               #  1500 Thr Phe Gln Gly Gln Pro Glu Arg Asp Glu Ly #s Ile Lys Glu Pro Thr 1505                1510 #                1515  #               1520 Leu Leu Gly Phe His Thr Ala Ser Gly Lys Ly #s Val Lys Ile Ala Lys                 1525  #               1530   #              1535 Glu Ser Leu Asp Lys Val Lys Asn Leu Phe As #p Glu Lys Glu Gln Gly             1540      #           1545       #          1550 Thr Ser Glu Ile Thr Ser Phe Ser His Gln Tr #p Ala Lys Thr Leu Lys         1555          #       1560           #      1565 Tyr Arg Glu Ala Cys Lys Asp Leu Glu Leu Al #a Cys Glu Thr Ile Glu     1570              #    1575              #   1580 Ile Thr Ala Ala Pro Lys Cys Lys Glu Met Gl #n Asn Ser Leu Asn Asn 1585                1590 #                1595  #               1600 Asp Lys Asn Leu Val Ser Ile Glu Thr Val Va #l Pro Pro Lys Leu Leu                 1605  #               1610   #              1615 Ser Asp Asn Leu Cys Arg Gln Thr Glu Asn Le #u Lys Thr Ser Lys Ser             1620      #           1625       #          1630 Ile Phe Leu Lys Val Lys Val His Glu Asn Va #l Glu Lys Glu Thr Ala         1635          #       1640           #      1645 Lys Ser Pro Ala Thr Cys Tyr Thr Asn Gln Se #r Pro Tyr Ser Val Ile     1650              #   1655               #  1660 Glu Asn Ser Ala Leu Ala Phe Tyr Thr Ser Cy #s Ser Arg Lys Thr Ser 1665                1670 #                1675  #               1680 Val Ser Gln Thr Ser Leu Leu Glu Ala Lys Ly #s Trp Leu Arg Glu Gly                 1685  #               1690   #              1695 Ile Phe Asp Gly Gln Pro Glu Arg Ile Asn Th #r Ala Asp Tyr Val Gly             1700      #           1705       #          1710 Asn Tyr Leu Tyr Glu Asn Asn Ser Asn Ser Th #r Ile Ala Glu Asn Asp         1715          #       1720           #      1725 Lys Asn His Leu Ser Glu Lys Gln Asp Thr Ty #r Leu Ser Asn Ser Ser     1730              #   1735               #  1740 Met Ser Asn Ser Tyr Ser Tyr His Ser Asp Gl #u Val Tyr Asn Asp Ser 1745                1750 #                1755  #               1760 Gly Tyr Leu Ser Lys Asn Lys Leu Asp Ser Gl #y Ile Glu Pro Val Leu                 1765  #               1770   #              1775 Lys Asn Val Glu Asp Gln Lys Asn Thr Ser Ph #e Ser Lys Val Ile Ser             1780      #           1785       #          1790 Asn Val Lys Asp Ala Asn Ala Tyr Pro Gln Th #r Val Asn Glu Asp Ile         1795          #       1800           #      1805 Cys Val Glu Glu Leu Val Thr Ser Ser Ser Pr #o Cys Lys Asn Lys Asn     1810              #   1815               #  1820 Ala Ala Ile Lys Leu Ser Ile Ser Asn Ser As #n Asn Phe Glu Val Gly 1825                1830 #                1835  #               1840 Pro Pro Ala Phe Arg Ile Ala Ser Gly Lys Il #e Arg Leu Cys Ser His                 1845  #               1850   #              1855 Glu Thr Ile Lys Lys Val Lys Asp Ile Phe Th #r Asp Ser Phe Ser Lys             1860      #           1865       #          1870 Val Ile Lys Glu Asn Asn Glu Asn Lys Ser Ly #s Ile Cys Gln Thr Lys         1875          #       1880           #      1885 Ile Met Ala Gly Cys Tyr Glu Ala Leu Asp As #p Ser Glu Asp Ile Leu     1890              #    1895              #   1900 His Asn Ser Leu Asp Asn Asp Glu Cys Ser Me #t His Ser His Lys Val 1905                1910 #                1915  #               1920 Phe Ala Asp Ile Gln Ser Glu Glu Ile Leu Gl #n His Asn Gln Asn Met                 1925  #               1930   #              1935 Ser Gly Leu Glu Lys Val Ser Lys Ile Ser Pr #o Cys Asp Val Ser Leu             1940      #           1945       #          1950 Glu Thr Ser Asp Ile Cys Lys Cys Ser Ile Gl #y Lys Leu His Lys Ser         1955          #       1960           #      1965 Val Ser Ser Ala Asn Thr Cys Gly Ile Phe Se #r Thr Ala Ser Gly Lys     1970              #   1975               #  1980 Ser Val Gln Val Ser Asp Ala Ser Leu Gln As #n Ala Arg Gln Val Phe 1985                1990 #                1995  #               2000 Ser Glu Ile Glu Asp Ser Thr Lys Gln Val Ph #e Ser Lys Val Leu Phe                 2005  #               2010   #              2015 Lys Ser Asn Glu His Ser Asp Gln Leu Thr Ar #g Glu Glu Asn Thr Ala             2020      #           2025       #          2030 Ile Arg Thr Pro Glu His Leu Ile Ser Gln Ly #s Gly Phe Ser Tyr Asn         2035          #       2040           #      2045 Val Val Asn Ser Ser Ala Phe Ser Gly Phe Se #r Thr Ala Ser Gly Lys     2050              #   2055               #  2060 Gln Val Ser Ile Leu Glu Ser Ser Leu His Ly #s Val Lys Gly Val Leu 2065                2070 #                2075  #               2080 Glu Glu Phe Asp Leu Ile Arg Thr Glu His Se #r Leu His Tyr Ser Pro                 2085  #               2090   #              2095 Thr Ser Arg Gln Asn Val Ser Lys Ile Leu Pr #o Arg Val Asp Lys Arg             2100      #           2105       #          2110 Asn Pro Glu His Cys Val Asn Ser Glu Met Gl #u Lys Thr Cys Ser Lys         2115          #       2120           #      2125 Glu Phe Lys Leu Ser Asn Asn Leu Asn Val Gl #u Gly Gly Ser Ser Glu     2130              #   2135               #  2140 Asn Asn His Ser Ile Lys Val Ser Pro Tyr Le #u Ser Gln Phe Gln Gln 2145                2150 #                2155  #               2160 Asp Lys Gln Gln Leu Val Leu Gly Thr Lys Va #l Ser Leu Val Glu Asn                 2165  #               2170   #              2175 Ile His Val Leu Gly Lys Glu Gln Ala Ser Pr #o Lys Asn Val Lys Met             2180      #           2185       #          2190 Glu Ile Gly Lys Thr Glu Thr Phe Ser Asp Va #l Pro Val Lys Thr Asn         2195          #       2200           #      2205 Ile Glu Val Cys Ser Thr Tyr Ser Lys Asp Se #r Glu Asn Tyr Phe Glu     2210              #   2215               #  2220 Thr Glu Ala Val Glu Ile Ala Lys Ala Phe Me #t Glu Asp Asp Glu Leu 2225                2230 #                2235  #               2240 Thr Asp Ser Lys Leu Pro Ser His Ala Thr Hi #s Ser Leu Phe Thr Cys                 2245  #               2250   #              2255 Pro Glu Asn Glu Glu Met Val Leu Ser Asn Se #r Arg Ile Gly Lys Arg             2260      #           2265       #          2270 Arg Gly Glu Pro Leu Ile Leu Val Gly Glu Pr #o Ser Ile Lys Arg Asn         2275          #       2280           #      2285 Leu Leu Asn Glu Phe Asp Arg Ile Ile Glu As #n Gln Glu Lys Ser Leu     2290              #   2295               #  2300 Lys Ala Ser Lys Ser Thr Pro Asp Gly Thr Il #e Lys Asp Arg Arg Leu 2305                2310 #                2315  #               2320 Phe Met His His Val Ser Leu Glu Pro Ile Th #r Cys Val Pro Phe Arg                 2325  #               2330   #              2335 Thr Thr Lys Glu Arg Gln Glu Ile Gln Asn Pr #o Asn Phe Thr Ala Pro             2340      #           2345       #          2350 Gly Gln Glu Phe Leu Ser Lys Ser His Leu Ty #r Glu His Leu Thr Leu         2355          #       2360           #      2365 Glu Lys Ser Ser Ser Asn Leu Ala Val Ser Gl #y His Pro Phe Tyr Gln     2370              #   2375               #  2380 Val Ser Ala Thr Arg Asn Glu Lys Met Arg Hi #s Leu Ile Thr Thr Gly 2385                2390 #                2395  #               2400 Arg Pro Thr Lys Val Phe Val Pro Pro Phe Ly #s Thr Lys Ser His Phe                 2405  #               2410   #              2415 His Arg Val Glu Gln Cys Val Arg Asn Ile As #n Leu Glu Glu Asn Arg             2420      #           2425       #          2430 Gln Lys Gln Asn Ile Asp Gly His Gly Ser As #p Asp Ser Lys Asn Lys         2435          #       2440           #      2445 Ile Asn Asp Asn Glu Ile His Gln Phe Asn Ly #s Asn Asn Ser Asn Gln     2450              #    2455              #   2460 Ala Ala Ala Val Thr Phe Thr Lys Cys Glu Gl #u Glu Pro Leu Asp Leu 2465                2470 #                2475  #               2480 Ile Thr Ser Leu Gln Asn Ala Arg Asp Ile Gl #n Asp Met Arg Ile Lys                 2485  #               2490   #              2495 Lys Lys Gln Arg Gln Arg Val Phe Pro Gln Pr #o Gly Ser Leu Tyr Leu             2500      #           2505       #          2510 Ala Lys Thr Ser Thr Leu Pro Arg Ile Ser Le #u Lys Ala Ala Val Gly         2515          #       2520           #      2525 Gly Gln Val Pro Ser Ala Cys Ser His Lys Gl #n Leu Tyr Thr Tyr Gly     2530              #   2535               #  2540 Val Ser Lys His Cys Ile Lys Ile Asn Ser Ly #s Asn Ala Glu Ser Phe 2545                2550 #                2555  #               2560 Gln Phe His Thr Glu Asp Tyr Phe Gly Lys Gl #u Ser Leu Trp Thr Gly                 2565  #               2570   #              2575 Lys Gly Ile Gln Leu Ala Asp Gly Gly Trp Le #u Ile Pro Ser Asn Asp             2580      #           2585       #          2590 Gly Lys Ala Gly Lys Glu Glu Phe Tyr Arg Al #a Leu Cys Asp Thr Pro         2595          #       2600           #      2605 Gly Val Asp Pro Lys Leu Ile Ser Arg Ile Tr #p Val Tyr Asn His Tyr     2610              #   2615               #  2620 Arg Trp Ile Ile Trp Lys Leu Ala Ala Met Gl #u Cys Ala Phe Pro Lys 2625                2630 #                2635  #               2640 Glu Phe Ala Asn Arg Cys Leu Ser Pro Glu Ar #g Val Leu Leu Gln Leu                 2645  #               2650   #              2655 Lys Tyr Arg Tyr Asp Thr Glu Ile Asp Arg Se #r Arg Arg Ser Ala Ile             2660      #           2665       #          2670 Lys Lys Ile Met Glu Arg Asp Asp Thr Ala Al #a Lys Thr Leu Val Leu         2675          #       2680           #      2685 Cys Val Ser Asp Ile Ile Ser Leu Ser Ala As #n Ile Ser Glu Thr Ser     2690              #   2695               #  2700 Ser Asn Lys Thr Ser Ser Ala Asp Thr Gln Ly #s Val Ala Ile Ile Glu 2705                2710 #                2715  #               2720 Leu Thr Asp Gly Trp Tyr Ala Val Lys Ala Gl #n Leu Asp Pro Pro Leu                 2725  #               2730   #              2735 Leu Ala Val Leu Lys Asn Gly Arg Leu Thr Va #l Gly Gln Lys Ile Ile             2740      #           2745       #          2750 Leu His Gly Ala Glu Leu Val Gly Ser Pro As #p Ala Cys Thr Pro Leu         2755          #       2760           #      2765 Glu Ala Pro Glu Ser Leu Met Leu Lys Ile Se #r Ala Asn Ser Thr Arg     2770              #   2775               #  2780 Pro Ala Arg Trp Tyr Thr Lys Leu Gly Phe Ph #e Pro Asp Pro Arg Pro 2785                2790 #                2795  #               2800 Phe Pro Leu Pro Leu Ser Ser Leu Phe Ser As #p Gly Gly Asn Val Gly                 2805  #               2810   #              2815 Cys Val Asp Val Ile Ile Gln Arg Ala Tyr Pr #o Ile Gln Arg Met Glu             2820      #           2825       #          2830 Lys Thr Ser Ser Gly Leu Tyr Ile Phe Arg As #n Glu Arg Glu Glu Glu         2835          #       2840           #      2845 Lys Glu Ala Ala Lys Tyr Val Glu Ala Gln Gl #n Lys Arg Leu Glu Ala     2850              #   2855               #  2860 Leu Phe Thr Lys Ile Gln Glu Glu Phe Glu Gl #u His Glu Glu Asn Thr 2865                2870 #                2875  #               2880 Thr Lys Pro Tyr Leu Pro Ser Arg Ala Leu Th #r Arg Gln Gln Val Arg                 2885  #               2890   #              2895 Ala Leu Gln Asp Gly Ala Glu Leu Tyr Glu Al #a Val Lys Asn Ala Ala             2900      #           2905       #          2910 Asp Pro Ala Tyr Leu Glu Gly Tyr Phe Ser Gl #u Glu Gln Leu Arg Ala         2915          #       2920           #      2925 Leu Asn Asn His Arg Gln Met Leu Asn Asp Ly #s Lys Gln Ala Gln Ile     2930              #   2935               #  2940 Gln Leu Glu Ile Arg Lys Ala Met Glu Ser Al #a Glu Gln Lys Glu Gln 2945                2950 #                2955  #               2960 Gly Leu Ser Arg Asp Val Thr Thr Val Trp Ly #s Leu Arg Ile Val Ser                 2965  #               2970   #              2975 Tyr Ser Lys Lys Glu Lys Asp Ser Val Ile Le #u Ser Ile Trp Arg Pro             2980      #           2985       #          2990 Ser Ser Asp Leu Tyr Ser Leu Leu Thr Glu Gl #y Lys Arg Tyr Arg Ile         2995          #       3000           #      3005 Tyr His Leu Ala Thr Ser Lys Ser Lys Ser Ly #s Ser Glu Arg Ala Asn     3010              #   3015               #  3020 Ile Gln Leu Ala Ala Thr Lys Lys Thr Gln Ty #r Gln Gln Leu Pro Val 3025                3030 #                3035  #               3040 Ser Asp Glu Ile Leu Phe Gln Ile Tyr Gln Pr #o Arg Glu Pro Leu His                 3045  #               3050   #              3055 Phe Ser Lys Phe Leu Asp Pro Asp Phe Gln Pr #o Ser Cys Ser Glu Val             3060      #           3065       #          3070 Asp Leu Ile Gly Phe Val Val Ser Val Val Ly #s Lys Thr Gly Leu Ala         3075          #       3080           #      3085 Pro Phe Val Tyr Leu Ser Asp Glu Cys Tyr As #n Leu Leu Ala Ile Lys     3090              #   3095               #  3100 Phe Trp Ile Asp Leu Asn Glu Asp Ile Ile Ly #s Pro His Met Leu Ile 3105                3110 #                3115  #               3120 Ala Ala Ser Asn Leu Gln Trp Arg Pro Glu Se #r Lys Ser Gly Leu Leu                 3125  #               3130   #              3135 Thr Leu Phe Ala Gly Asp Phe Ser Val Phe Se #r Ala Ser Pro Lys Glu             3140      #           3145       #          3150 Gly His Phe Gln Glu Thr Phe Asn Lys Met Ly #s Asn Thr Val Glu Asn         3155          #       3160           #      3165 Ile Asp Ile Leu Cys Asn Glu Ala Glu Asn Ly #s Leu Met His Ile Leu     3170              #   3175               #  3180 His Ala Asn Asp Pro Lys Trp Ser Thr Pro Th #r Lys Asp Cys Thr Ser 3185                3190 #                3195  #               3200 Gly Pro Tyr Thr Ala Gln Ile Ile Pro Gly Th #r Gly Asn Lys Leu Leu                 3205  #               3210   #              3215 Met Ser Ser Pro Asn Cys Glu Ile Tyr Tyr Gl #n Ser Pro Leu Ser Leu             3220      #           3225       #          3230 Cys Met Ala Lys Arg Lys Ser Val Ser Thr Pr #o Val Ser Ala Gln Met         3235          #       3240           #      3245 Thr Ser Lys Ser Cys Lys Gly Glu Lys Glu Il #e Asp Asp Gln Lys Asn     3250              #   3255               #  3260 Cys Lys Lys Arg Arg Ala Leu Asp Phe Leu Se #r Arg Leu Pro Leu Pro 3265                3270 #                3275  #               3280 Pro Pro Val Ser Pro Ile Cys Thr Phe Val Se #r Pro Ala Ala Gln Lys                 3285  #               3290   #              3295 Ala Phe Gln Pro Pro Arg Ser Cys Gly Thr Ly #s Tyr Glu Thr Pro Ile             3300      #           3305       #          3310 Lys Lys Lys Glu Leu Asn Ser Pro Gln Met Th #r Pro Phe Lys Lys Phe         3315          #       3320           #      3325 Asn Glu Ile Ser Leu Leu Glu Ser Asn Ser Il #e Ala Asp Glu Glu Leu     3330              #   3335               #  3340 Ala Leu Ile Asn Thr Gln Ala Leu Leu Ser Gl #y Ser Thr Gly Glu Lys 3345                3350 #                3355  #               3360 Gln Phe Ile Ser Val Ser Glu Ser Thr Arg Th #r Ala Pro Thr Ser Ser                 3365  #               3370   #              3375 Glu Asp Tyr Leu Arg Leu Lys Arg Arg Cys Th #r Thr Ser Leu Ile Lys             3380      #           3385       #          3390 Glu Gln Glu Ser Ser Gln Ala Ser Thr Glu Gl #u Cys Glu Lys Asn Lys         3395          #       3400           #      3405 Gln Asp Thr Ile Thr Thr Lys Lys Tyr Ile     3410              #  3415 (2) INFORMATION FOR SEQ ID NO:5:      (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 10           (B) TYPE: amino acid           (C) STRANDEDNESS: single           (D) TOPOLOGY: unknown     (ix) FEATURE:           (A) NAME/KEY: Granin Co #nsensus Sequence     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: #5: Glu Asn Leu Ser Xaa Xaa Asp Xaa Glu Leu 1               5    #                10 (2) INFORMATION FOR SEQ ID NO:6:      (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 10           (B) TYPE: amino acid           (C) STRANDEDNESS: single           (D) TOPOLOGY: unknown     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: #6: Glu Asn Leu Ser Ser Glu Asp Glu Glu Leu 1               5    #                10 (2) INFORMATION FOR SEQ ID NO:7:      (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 10           (B) TYPE: amino acid           (C) STRANDEDNESS: single           (D) TOPOLOGY: unknown     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: #7: Glu Asn Leu Ser Ser Glu Asp Glu Glu Leu 1               5    #                10 (2) INFORMATION FOR SEQ ID NO:8:      (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 10           (B) TYPE: amino acid           (C) STRANDEDNESS: single           (D) TOPOLOGY: unknown     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: #8: Glu Ser Asp Ser Thr Glu Asp Glu Asp Leu 1               5    #                10 (2) INFORMATION FOR SEQ ID NO:9:      (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 10           (B) TYPE: amino acid           (C) STRANDEDNESS: single           (D) TOPOLOGY: unknown     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: #9: Glu Ser Asn Ser Ile Ala Asp Glu Glu Leu 1             5      #           10 (2) INFORMATION FOR SEQ ID NO:10:      (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 10           (B) TYPE: amino acid           (C) STRANDEDNESS: single           (D) TOPOLOGY: unknown     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: #10: Glu Ser Leu Ser Ala Ile Glu Ala Glu Leu 1               5    #                10 (2) INFORMATION FOR SEQ ID NO:11:      (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 10           (B) TYPE: amino acid           (C) STRANDEDNESS: single           (D) TOPOLOGY: unknown     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: #11: Glu Ser Leu Ser Ala Ile Glu Ala Glu Leu 1               5    #                10 (2) INFORMATION FOR SEQ ID NO:12:      (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 10           (B) TYPE: amino acid           (C) STRANDEDNESS: single           (D) TOPOLOGY: unknown     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: #12: Glu Ser Leu Ser Ala Ile Glu Ala Glu Leu 1               5    #                10 (2) INFORMATION FOR SEQ ID NO:13:      (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 10           (B) TYPE: amino acid           (C) STRANDEDNESS: single           (D) TOPOLOGY: unknown     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: #13: Glu Ser Leu Ser Ala Ile Glu Ala Glu Leu 1               5    #                10 (2) INFORMATION FOR SEQ ID NO:14:      (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 10           (B) TYPE: amino acid           (C) STRANDEDNESS: single           (D) TOPOLOGY: unknown     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: #14: Glu Asn Leu Ala Ala Met Asp Leu Glu Leu 1               5    #                10 (2) INFORMATION FOR SEQ ID NO:15:      (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 10           (B) TYPE: amino acid           (C) STRANDEDNESS: single           (D) TOPOLOGY: unknown     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: #15: Glu Asn Leu Ala Ala Met Asp Leu Glu Leu 1               5    #                10 (2) INFORMATION FOR SEQ ID NO:16:      (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 10           (B) TYPE: amino acid           (C) STRANDEDNESS: single           (D) TOPOLOGY: unknown     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: #16: Glu Asn Leu Ala Ala Met Asp Leu Glu Leu 1               5    #                10 (2) INFORMATION FOR SEQ ID NO:17:      (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 10           (B) TYPE: amino acid           (C) STRANDEDNESS: single           (D) TOPOLOGY: unknown     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: #17: Glu Asn Leu Asn Asp Lys Asp Gln Glu Leu 1               5    #                10 (2) INFORMATION FOR SEQ ID NO:18:      (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 10           (B) TYPE: amino acid           (C) STRANDEDNESS: single           (D) TOPOLOGY: unknown     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: #18: Glu Asn Leu Asn Asp Lys Asp Gln Glu Leu 1               5    #                10 (2) INFORMATION FOR SEQ ID NO:19:      (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 10           (B) TYPE: amino acid           (C) STRANDEDNESS: single           (D) TOPOLOGY: unknown     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: #19: Asp Asn Leu Asn Asp Lys Asp Gln Glu Leu 1               5    #                10 (2) INFORMATION FOR SEQ ID NO:20:      (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 10           (B) TYPE: amino acid           (C) STRANDEDNESS: single           (D) TOPOLOGY: unknown     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: #20: Glu Asn Leu Asn Xaa Xaa Asp Gln Glu Leu 1               5    #                10 (2) INFORMATION FOR SEQ ID NO:21:      (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 10           (B) TYPE:  amino aci #d           (C) STRANDEDNESS:  sing #le           (D) TOPOLOGY: unknown     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: #21: Glu Asn Leu Asp Glu Thr Ile Ala Leu Gln 1               5    #                10 (2) INFORMATION FOR SEQ ID NO:22:      (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 10           (B) TYPE:  amino aci #d           (C) STRANDEDNESS:  sing #le           (D) TOPOLOGY: unknown     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: #22: Glu Asn Leu Asp Glu Thr Ile Ala Leu Gln 1               5    #                10 (2) INFORMATION FOR SEQ ID NO:23:      (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 10           (B) TYPE:  amino aci #d           (C) STRANDEDNESS:  sing #le           (D) TOPOLOGY: unknown     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: #23: Gly Asn Ile Pro Asn Ile Val Ala Glu Leu 1               5    #                10 (2) INFORMATION FOR SEQ ID NO:24:      (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 10           (B) TYPE:  amino aci #d           (C) STRANDEDNESS:  sing #le           (D) TOPOLOGY: unknown     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: #24: Gly Asn Ile Pro Asn Ile Val Ala Glu Leu 1               5    #                10 (2) INFORMATION FOR SEQ ID NO:25:      (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 10           (B) TYPE:  amino aci #d           (C) STRANDEDNESS:  sing #le           (D) TOPOLOGY: unknown     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: #25: Gly Asn Ile Pro Asn Ile Val Ala Glu Leu 1               5    #                10 (2) INFORMATION FOR SEQ ID NO:26:      (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 10           (B) TYPE:  amino aci #d           (C) STRANDEDNESS:  sing #le           (D) TOPOLOGY: unknown     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: #26: Gly Asn Ile Pro Asn Ile Val Ala Glu Leu 1               5    #                10 

What is claimed is:
 1. A method to reduce the growth of a prostate tumor in a mammal, comprising: injecting into said prostate tumor a retroviral construct comprising a BRCA1 nucleic acid sequence operably linked to a promoter and encoding a BRCA1 polypeptide having tumor suppressor activity, wherein said BRCA1 polypeptide is expressed in said prostate tumor at a level and for a period of time sufficient to reduce the growth of said prostate tumor.
 2. The method of claim 1, wherein said tumor is gene-linked hereditary prostate cancer.
 3. The method of claim 1, wherein said tumor is sporadic prostate cancer.
 4. The method of claim 1, wherein said BRCA1 polypeptide is a wild type BRCA1 polypeptide.
 5. The method of claim 4, wherein the retroviral construct is an LXSN retroviral construct.
 6. The method of claim 4, wherein said BRCAI nucleic acid sequence encoding said BRCA1 polypeptide is selected from the group consisting of: (a) a nucleic acid sequence as set forth in SEQ ID NO: 1; and (b) a nucleic acid sequence encoding a polypeptide as set forth in SEQ ID NO:2.
 7. The method of claim 4, wherein said tumor is gene-linked hereditary prostate cancer.
 8. The method of claim 4, wherein said tumor is sporadic prostate cancer. 